Dithioamine reducing agents

ABSTRACT

Dithioamine reducing agents useful for the reduction of disulfide bonds. The reducing agents of this invention are useful, for example, to reduce disulfide bonds, particularly in proteins, or to prevent the formation of disulfide bonds, particularly in proteins and other biological molecules. Reducing agents of this invention are useful and suitable for application in a variety of biological applications, particularly as research and synthetic reagents. The invention provides S-acylated dithioamines which can be selectively activated reducing agents by removal of the S-acyl groups enzymatically or chemically. The invention further provides dithiane precursors of thioamino reducing agents. The invention provides dithioamine reducing agents, S-acylated dithioamines and dithianes which are immobilized on surfaces, including among others, glass, quartz, microparticles, nanoparticles and resins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application61/599,380, filed Feb. 15, 2012, which is incorporated by referenceherein in its entirety.

STATEMENT REGARDING GOVERNMENT FUNDING

This invention was made with government support under GM044783 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Approximately 20% of human proteins are predicted to contain disulfidebonds between cysteine residues. [1] Small-molecule thiols can reducethese (and other) disulfide bonds, thereby modulating biomolecularfunction. [2] The reaction mechanism involves thiol-disulfideinterchange initiated by a thiolate. [3] The ensuing mixed disulfide,however, can become trapped if the reagent is a monothiol, such asβ-mercaptoethanol (βME). [4]

To overcome this problem, Cleland developed racemic(2S,3S)-1,4-dimercaptobutane-2,3-diol (dithiothreitol or DTT; Table 1),a dithiol that resolves a mixed disulfide by forming a six-memberedring. [2a, 5] DTT is a potent reducing agent (E°′—0.327 V) [2g] and hasbeen, despite its high cost, the preferred reagent for the quantitativereduction of disulfide bonds and is now the standard reagent forreducing disulfide bonds in biological molecules. [6, 7] Atphysiological pH, DTT is, however, a sluggish reducing agent. Thereactivity of a dithiol is governed by the lower of its two thiol pK_(a)values. [2, 3] With its lower thiol pK_(a) value being 9.2, [Table 1]greater than 99% of DTT thiol groups are protonated at pH 7 and thusunreactive (i.e., less than 1% of DTT residues are in the reactivethiolate form at pH 7 [8])

Thus, there is a need in the art for reducing agents useful inbiological systems, for example, for the reduction of disulfide bonds,which exhibit properties improved over those of prior art reducingagents. The present invention provides dithiol amines which can beprepared from inexpensive starting materials in high yield and whichexhibit desirable improved properties as reducing agents.

SUMMARY OF THE INVENTION

The present invention provides improved dithioamine reducing agentsuseful, in particular, for the reduction of disulfide bonds. Thereducing agents of this invention are useful, for example, to reducedisulfide bonds, particularly in proteins, or to prevent the formationof disulfide bonds, particularly in proteins and other biologicalmolecules (e.g., thiolated species, such as thiolated nucleic acids).Reducing agents of this invention can be employed to regulate proteinfunction in proteins in which a sulfhydryl group (such as those ofcysteine residues) is associated with biological activity. Reducingagents of this invention can prevent inactivation of a given protein orenhance activation of a given protein or other biological molecule invitro and/or in vivo. Reducing agents of this invention can prevent orreduce oxidation of cysteine residues in proteins and prevent theformation of reduced activity protein dimers (or other oligomers).Reducing agents of this invention are useful and suitable forapplication in a variety of biological applications, particularly asresearch and synthetic reagents. In specific embodiments, the inventionprovides S-acylated dithioamines which can be selectively activated asreducing agents by removal of the S-acyl groups enzymatically orchemically. In specific embodiments, the invention provides dithioaminereducing agents and S-acylated dithioamines which are immobilized onsurfaces, including among others, glass, quartz, microparticles andnanoparticles.

In specific embodiments, dithioamine reducing agents of this inventionexhibit a thiol pK_(a) value less than 9.2, preferably less than 9.0 andmore preferably less than 8.5. In specific embodiments, dithioaminereducing agents of this invention exhibit disulfide reduction potentialmore negative than −0.28 V, preferably more negative than −0.30 V andmore preferably more negative than −0.32 V. In specific embodiments, thedithiol reducing agent contains an amine-containing group substituted ona carbon alpha to the carbon upon which a thiol is substituted. In aspecific embodiment, the pK_(a) of the amine containing group which issubstituted on a carbon alpha to a carbon upon which a thiol issubstituted is greater than the pK_(a) of the thiol groups in thereducing agent. In a specific embodiment, the pK_(a) of thisamine-containing group is 10 or greater. In a more specific embodiment,the pK_(a) of this amine-containing group is 10.5 or greater.

Reducing agents of this invention include compounds of formula I andsalts thereof:

where:

R₁ is hydrogen, or an unsubstituted alkyl group having 1 to 3 carbonatoms; each R₂ and R₃ is independently hydrogen, an alkyl group having1-3 carbon atoms, a phenyl or a benzyl group, wherein each alkyl,phenyl, or benzyl group is optionally substituted with one or morenon-hydrogen substituents;

each R₄ and R₅ is independently hydrogen, a halogen, a cyano group, anitro group, a hydroxyl, an alkyl group having 1-6 carbon atoms, aphenyl, a benzyl group, an —N(R₉)₂, or a —COR₁₀ group, wherein eachalkyl, phenyl, or benzyl group is optionally substituted with one ormore of non-hydrogen substituents;

each R₆ and R₇ is independently hydrogen, a 1-12 carbon alkyl group, anaryl group, a heterocyclic group, a heteroaryl group, a —COR₁₁ group, a—CO—NHR₁₁ group, a —CO—NHR₁₁ group, a —SO₂—R₁₁ group, or a—(CH₂)_(n)—R₁₂ group, wherein each alkyl, aryl heterocyclic orheteroaryl group is optionally substituted with one or more non-hydrogensubstituents;

each R₈ is independently hydrogen or an acyl group (—CO—R₁₃), wherein:

each R₉ is independently hydrogen, an alkyl group having 1-12 carbonatoms, an aryl group, a heterocyclic group, a heteroaryl group, a —COR₁₁group, a —COOR₁₁ group, a —CO—NHR₁₁ group, a —CO—NHR₁₁ group, a —SO₂—R₁₁group, or a —(CH₂)_(n)—R₁₂ group, where n is an integer ranging from1-12, wherein each alkyl, aryl, heterocyclic or heteroaryl group isoptionally substituted with one or more non-hydrogen substituents;

each R₁₀ is independently hydrogen, an alkyl group having 1-12 carbonatoms, a phenyl or benzyl group, wherein each alkyl, phenyl or benzylgroup is optionally substituted with one or more of non-hydrogensubstituents;

each R₁₁ and R₁₂ is independently hydrogen, an alkyl group having 1-12carbon atoms, an aryl group, a heterocyclic group, a heteroaryl group, a-L-T group or a -M group, wherein each alkyl, aryl, heterocyclic orheteroaryl group is optionally substituted with one or more non-hydrogensubstituents; -L- is a divalent linker group and T is a biologicalspecies or a surface to which the reducing agent is linked; and -M is areactive group or a spacer moiety carrying a reactive group; and

each R₁₃ is independently hydrogen, an alkyl group having 1-12 carbonatoms, an aryl group, a heterocyclic group, or a heteroaryl group,wherein each alkyl, aryl heterocyclic or heteroaryl group is optionallysubstituted with one or more non-hydrogen substituents

In specific embodiments, the —NR₆R₇ group retains positive charge underapplication conditions. In specific embodiments, application conditionsinclude use in vivo or in vitro at a pH between 5.5-8.5, between 6 to 8,between 7 to 8, or between 7.2 to 7.6. In specific embodiments, the—NR₆R₇ group is protonated under application conditions. Retention of apositive charge on this nitrogen is believed to be beneficial toactivity of the reducing agent. In specific embodiments, both of R₆ andR₇ are groups other than hydrogen, e.g., one of R₆ or R₇ is an alkylgroup and one or R₆ or R₇ is an —COR₁₃ group as defined above.

In a specific embodiment, dithioamine compounds of the invention includecompounds of formula IA and salts thereof:

where the optical configuration at the indicated chiral center is asindicated in the formula and variables are as defined above.

Compounds of the invention also include those of formula IB and saltsthereof:

where the optical configuration at the indicated chiral center is asindicated in the formula and variables are as defined above.

In specific embodiments of formulas IA and IB, all of R₁-R₈ arehydrogens and the compounds are S-2-amino-1,4-dimercaptobutane(S-dithiobutylamine, S-DTBA) or R-2-amino-1,4-dimercaptobutane(R-dithiobutylamine, R-DTBA) or salts thereof, such as the hydrochloridesalts thereof.

The invention further relates to the dithiane compounds and saltsthereof of formula II which are the oxidized form of the dithioamines:

where R₁-R₇ are as defined above for formula I. In specific embodiments,dithianes of the invention can have the specific optical configurationat the carbon substituted with —NR₆R₇ as indicated in formulas IA andIB. In a specific embodiment, dithiane compounds of the inventioninclude the compounds of formula II, with the exception of the compoundof formula II, wherein all of R₁-R₇ are hydrogens. The dithianes areuseful, for example, in the preparation of dithioamine reducing agentsand particularly in the preparation of immobilized dithioamine reducingagents.

The invention further provides methods for preventing or reducing theoxidation of one or more sulfhydryl groups in a biological molecule,particularly a peptide or protein, in vivo or in vitro by contacting thebiological molecule with one or more dithioamine compounds of formulasI, IA or IB. In a specific embodiment, an S-acylated dithioamine offormula I, IA or IB is employed and is activated chemically orenzymatically by removal of a S-acyl group prior to or at about the sametime as the protein is contacted. In specific embodiments, the inventionprovides a method for preventing or reducing the formation of disulfidebonds or for cleaving already-formed disulfide bonds in or between oneor more molecules containing sulfhydryl groups or disulfide bonds bycontacting the one or more molecules with one or more dithioaminecompounds of formulas I, IA or IB. In a specific embodiment, anS-acylated dithioamine of formula I, IA or IB is employed and isactivated chemically or enzymatically by removal of a S-acyl group priorto or at about the same time as the one or more molecules are contacted.

In a more specific embodiment, the invention provides a method ofregulating a biological activity of a protein wherein said biologicalactivity is associated with the presence or absence of a sulfhydrylgroup or the formation or cleavage of a disulfide bond. In this method,a dithioamine of this invention is employed to prevent or reduce theoxidation of one or more sulfhydryl groups in a protein or to prevent orreduce the formation of a disulfide bond or to cleave an already-formeddisulfide bond.

The invention further relates to reagent kits which comprise one or moredithioamines of formulas I, IA, or IB individually packaged therein inselected amounts for use as a reducing agent. More specifically, suchkits are for preventing or reducing disulfide bond formation or forcleaving disulfide bonds. Reagent kits may further comprise one or moresolvents or other reagents for carrying out a reduction.

Additional embodiments of the invention will be apparent from a reviewof the drawings, detailed description and the examples herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B are graphs illustrating the time-course for the reductionof a mixed disulfide in exemplary small molecules by DTBA and DTT in 50mM potassium phosphate buffer. FIG. 1A shows reduction of oxidized βME(β-mercaptoethanol) where k_(obs) ^(DTBA)/k_(obs) ^(DTT)=3.5 at pH 7.0;k_(obs) ^(DTBA)/k_(obs) ^(DTT)=4.4 at pH 5.5. FIG. 1B shows reduction ofoxidized I-glutathione; k_(obs) ^(DTBA)/k_(obs) ^(DTT)=5.2 at pH 7.0.

FIGS. 2A and B are grpahs showing the time-course for the reduction of amixed disulfide in exemplary enzymic active sites by DTBA and DTT in0.10 M imidazole-HCl buffer, pH 7.0, containing EDTA (2 mM). FIG. 2Ashows a reduction of papain-Cys35-S—S—CH₃, where k_(obs) ^(DTBA)/k_(obs)^(DTT)=14. FIG. 2B shows reduction of creatinekinase-Cys283-S—S—I-glutathione; k_(obs) ^(DTBA/k) _(obs) ^(DTT)=1.1.

FIG. 3 is a graph showing the effect of pH on absorbance at 238 nm(A₂₃₈) of DTBA (0.10 mM) in 0.10 M potassium phosphate buffer. Fittingthe data to eq 1 yielded pK_(a) values of 8.2±0.2 and 9.3±0.1, andextinction coefficients of ε_(SH) ^(SH)=83.27 M⁻¹ cm⁻¹, ε_(SH)^(S−)=3436 M⁻¹ cm⁻¹, and ε_(S−) ^(S−)=M⁻¹ cm⁻¹ with r²>0.99.

FIG. 4 illustrates a representative HPLC chromatogram of the redoxequilibrium between DTBA and DTT. Compounds were detected by theirabsorbance at 205 nm.

FIG. 5 illustrates a representative HPLC chromatogram of the redoxequilibrium between BMS and DTT. Compounds were detected by theirabsorbance at 205 nm.

FIG. 6 illustrates the ultraviolet spectrum of oxidized DTBA andoxidized DTT in DPBS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based at least in part on the finding that DTBA(specifically S-DTBA) has thiol pK_(a) values that are significantlylower than those of DTT. DTBA is a non-racemic dithiol with low thiolpK_(a) and disulfide E°′. Additionally DTBA and various derivativesthereof can be prepared from inexpensive sources. DTBA in particular canbe prepared in high yield from inexpensive aspartic acid. Various DTBAderivatives can be prepared from derivatives of aspartic acid. Theinitial target (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine orDTBA; Table 1) is synthesized from L-aspartic acid, which is an abundantamino acid, see Scheme 1. [9, 10] Salts of the compounds of formulas I,IA and IB can be prepared by art-known methods as illustrated in theexamples herein.

The invention provides dithioamine reducing agents of formulas I, IA andIB:

where variables R₁-R₈ are as defined above and further defined below.

In specific embodiments, the invention provides reducing agents offormulas I, IA and IB which exhibit a thiol pK_(a) value less than 9.2,preferably less than 9.0 and more preferably less than 8.5. In specificembodiments, the invention provides reducing agents of formulas I, IA orIB which in addition exhibit disulfide reduction potential more negativethan −0.28 V, preferably more negative than −0.30 V and more preferablymore negative than −0.32 V. In a specific embodiment, the inventionprovides reducing agents of formulas I, IA and IB in which the pK_(a) ofthe —NR₆R₇ group is greater than the pK_(a) of the thiol groups in thereducing agent. In a specific embodiment, the pK_(a) of this —NR₆R₇group is 10 or greater. In a more specific embodiment, the pK_(a) ofthis —NR₆R₇ group is 10.5 or greater.

In specific embodiments, when one or more of R₂-R₅ are an alkyl grouphaving 1-3 carbon atoms, a phenyl or a benzyl group, each alkyl, phenyl,or benzyl group is unsubstituted or is substituted with one or morenon-hydrogen substituents selected from substituents W₂. In specificembodiments, when R₁₀ is an alkyl group having 1-12 carbon atoms, aphenyl or benzyl group, each alkyl, phenyl or benzyl group isunsubstituted or is substituted with one or more non-hydrogensubstituents selected from substituents W₂.

Substituents W₂ are one or more substituents selected from: halogen, anoxo group (═O), cyano group, a nitro group, a hydroxyl, an unsubstitutedalkyl group having 1-3 carbon atoms, a halogen-substituted alkyl grouphaving 1-3 carbon atoms, or an unsubstituted alkoxy group having 1-3carbon atoms.

Preferred W₂ substituents are one or more halogen, unsubstituted alkylor unsubstituted alkoxy. Specific W₂ substituents include one or more—CH₃, —C₂H₅, —CF₃, —COH, —COCH₃, —F, —Cl, —OCH₃, or —OC₂H₅. SubstituentsW₂ includes one, two or three of the listed substituents. In specificembodiments, R₂-R₅ are not substituted with nitro groups.

In specific embodiments, when one or more of R₆, R₇, R₉, R₁₁, R₁₂ or R₁₃is a 1-12 carbon alkyl group, an aryl group, a heterocyclic group, or aheteroaryl group, each alkyl, aryl heterocyclic or heteroaryl group isunsubstituted or is optionally substituted with one or more substituentsW_(3.)

Substituents W₃ are one or more substituents selected from:

-   halogen,-   an oxo group (═O),-   cyano group,-   nitro group,-   hydroxyl,-   an optionally substituted alkyl group having 1-6 carbon atoms,-   an unsubstituted alkyl group having 1-6 carbon atoms,-   a hydroxyl-substituted alkyl group having 1-6 carbon atoms,-   a halogen-substituted alkyl group having 1-6 carbon atoms,-   an unsubstituted alkoxy group having 1-6 carbon atoms,-   an alkyl group having 2-6 carbon atoms,-   an alkyenyl group having 2-6 carbon atoms,-   a 3-6-member alicyclic ring, wherein one or two ring carbons are    optionally replaced with —CO— and which may contain one or two    double bonds,-   an aryl group having 6-14 carbon ring atoms,-   a phenyl group,-   a benzyl group,-   a 5- or 6-member ring heterocyclic group having 1-3 heteroatoms    wherein one or two ring carbons are optionally replaced with —CO—    and which may contain one or two double bonds,-   a heteroaryl group having 1-3 heteroatoms (N, O or S),-   a —CO₂R₁₄ group,-   a —CON(R₁₅)₂ group,-   a —OCON(R₁₅)₂ group,-   a —N(R₁₅)₂ group,-   a —SO₂—OR₁₅ group,-   a —(CH₂)_(m)—OR₁₄ group, or-   a —(CH₂)_(m)—N(R₁₅)₂ group,-   where m is 1-8,

each R₁₄ is hydrogen; an unsubstituted alkyl group having 1-6 carbonatoms; an unsubstituted aryl group having 6-14 carbon atoms; anunsubstituted phenyl group; an unsubstituted benzyl group; anunsubstituted 5- or 6-member ring heterocyclic group, having 1-3heteroatoms and wherein one or two ring carbons are optionally replacedwith —CO— and which may contain one or two double bonds; or aunsubstituted heteroaryl group having 1-3 heteroatoms (N, O or S); and

-   each R₁₅ is hydrogen; an unsubstituted alkyl group having 1-6 carbon    atoms; an unsubstituted aryl group having 6-14 carbon atoms; an    unsubstituted phenyl group; an unsubstituted benzyl group; an    unsubstituted 5- or 6-member ring heterocyclic group, having 1-3    heteroatoms and wherein one or two ring carbons are optionally    replaced with —CO— and which may contain one or two double bonds; or    a unsubstituted heteroaryl group having 1-3 heteroatoms (N, O or S).

Preferred W₃ substituents are halogen, hydroxyl, oxo group (═O), a cyanogroup, a nitro group, unsubstituted and substituted alkyl groups asdefined above, unsubstituted alkoxy, unsubstituted phenyl or benzyl, andhalogen-substituted phenyl or benzyl. Specific W₂ substituents includealkyl groups having 1-3 carbon atoms, alkoxy groups having 1-3 carbonatoms, —CH₃, —C₂H₅, —CF₃, —COH, —COCH₃, —F, —Cl, —OCH₃, and —OC₂H₅. Inspecific embodiments, R₆, R₇, R₉, R₁₁, R₁₂ and R₁₃ are not substitutedwith nitro groups.

In a specific embodiment, dithioamine compounds of the invention includethe compound of formula I wherein all of R₁-R₈ are hydrogens,2-amino-1,4-dimercaptobutane or dithiobutylamine (DTBA) or a salt, suchas the hydrochloride salt, thereof.

In specific embodiments, the invention provided S-acylated dithioaminesof formulas IC and ID salts thereof and various optical isomers thereof:

where R₁-R₈ and R₁₃ are as defined above and herein below. TheseS-acylated dithiomaines are precursors of dithiomaine reducing agents ofthis invention. In specific embodiments, R₁₃ is an optionallysubstituted alkyl group having 1-6 carbon atoms, or a an optionallysubstituted phenyl or benzyl group. In specific embodiments, R₁₃ is anunsubstituted, straight-chain alkyl group having 1-6 carbon atoms or 1-3carbon atoms.

In specific embodiments, R₁₃ is an unsubstituted, branched alkyl grouphaving 3-6 carbon atoms. In specific embodiments, R₁₃ is ahydroxy-substituted alkyl group having 1-6 carbon atoms. In specificembodiments, R₁₃ is a butyl group, including all isomers thereof. Inspecific embodiments, R₁₃ is a t-butyl or neopentyl group. In specificembodiments, R₁₃ is an unsubstituted phenyl or benzyl group.

Compounds of formulas I, and IA-ID and II include all enantiomers anddiastereomers thereof.

In specific embodiments of formulas I, IA and IB each R₈ is hydrogen. Inspecific embodiments of formulas I, IA, IB and II, R₁ is hydrogen. Inspecific embodiments of formulas I, IA, IB and II, each R₂ and R₃ isindependently hydrogen or unsubstituted alkyl groups having 1-3 carbonatoms. In specific embodiments of formulas I, IA, IB and II, each R₂ andR₃ is a hydrogen. In specific embodiments of formulas I, IA, IB and II,each R₄ and R₅ is independently hydrogen, a halogen, a cyano group, anitro group, a hydroxyl, an alkyl group having 1-6 carbon atomsoptionally substituted with one or more halogens, or a —COR₁₀ groupwherein R₁₀ is hydrogen or an alkyl group having 1-6 carbon atomsoptionally substituted with one or more halogens. In specificembodiments, R₄ and R₅ are not nitro groups. In specific embodiments, R₄and R₅ are both hydrogens.

In specific embodiments of formulas I, IA, IB and II, R₆ and R₇ arehydrogens or unsubstituted alkyl groups having 1-6 carbon atoms. Inspecific embodiments of formulas I, IA, IB and II, R₆ and R₇ are bothhydrogens. In specific embodiments of formulas I, IA, IB and II, one ofR₆ and R₇ is a hydrogen. In specific embodiments, one of R₆ and R₇ is ahydrogen, and the other is a 1-12 carbon alkyl group, an aryl group, aheterocyclic group, a heteroaryl group, a —COR₁₁ group or a—(CH₂)_(n)—R₁₂ group. In specific embodiments, one of R₆ and R₇ is a—COR₁₁ group, a —CO—NHR₁₁ group, a —CO—NHR₁₁ group, a —SO₂—R₁₁ group, ora —(CH₂)_(n)—R₁₂ group, wherein R₁₁ or R₁₂ is a -L-T group or a -Mgroup.

In specific embodiments, the dithioamine of this invention issubstituted with a reactive group which allows its coupling to abiological species or to a surface either directly or indirectly througha spacer moiety. In specific embodiments, the reactive group is a latentreactive group, such as a protected group which can be selectivelyactivated for reaction for coupling of the compound directly orindirectly via a linker or spacer group to a T group or a surface. Inspecific embodiments, -M is or carries a reactive group or latentreactive group which reacts or can be activated (e.g., by deprotection)to react with one or more of an amine group, a carboxylic acid group, asulfhydryl group, a hydroxyl group, an aldehyde or ketone group, anazide group, an activated ester group, a thioester group, or aphosphinothioester group or reacts with one reactive group of ahomobifunctional or a heterobifunctional crosslinking reagent. Inspecific embodiments, when the reactive group is a sulfhydryl group,both of R₈ are acyl groups.

In specific embodiments, reactive groups are latent reactive groupswhich are protected amine groups, protected carboxylic acid groups,protected sulfhydryl groups, protected hydroxyl groups, or protectedaldehyde or ketone groups. Protective groups, for various reactivegroups are known in the art, for example as described in Wutts, P. G.and Greene, T. (2007) Green's Protecting Groups in Organic Synthesis(Fourth Edition) John Wiley & Sons, N.Y. This reference is incorporatedby reference herein for its description of protective groups for a givenreactive group and for methods of protecting reactive groups and methodsfor removing such protective groups. One or ordinary skill in the artcan select from among known alternatives a protective group appropriatefor a given reactive group under given conditions.

Amine-protective groups include among others: t-butyloxycarbo(BOC),9-fluorenylmethyloxycarbonyl (FMOC), acetyl, benzyl, carbamate,p-methoxyphenyl, tosyl, 4-nitrophenylsulfonyl, or 4-aminophenylsulfonyl. Carboxylic acid-protective groups include among others, esters(e.g., alkyl or aryl esters of the carboxylic acid), silyl esters, ororthoesters. Hydroxyl-protecting groups include among others, acylgroups (e.g., acetyl, benzoyl), beta-methoxyethoxymethyl ether,dimethoxytrityl, methoxytrityl, methoxymethyl ether, p-methoxybenzylether, pivaloyl, silyl ethers, methyl ethers or ethoxyethyl ethers.

In specific embodiments, the dithioamine of this invention isimmobilized onto a surface via a linker group -L-. In specificembodiments, the dithioamine of this invention is an S-acylateddithioamine of this invention immobilized onto a surface via a linkergroup -L-. In specific embodiments, the dithioamine of this invention isconjugated to a biological molecule via a linker group -L-. In specificembodiments, the dithioamine of this invention is an S-acylateddithioamine of this invention conjugated to a biological molecule via alinker group -L-. In specific embodiments, the biological molecule is apeptide or protein, a carbohydrate or a nucleic acid. In specificembodiments, the biological molecule is a ligand or substrate that bindsto a biological molecule, particularly where the biological molecule isa peptide or protein.

The invention provides dithioamine reducing agents immobilized onsurfaces. Such immobilized reducing agents can be used as recognized inthe art to reduce all types of disulfides, particularly biologicaldisulfides, including those in or between proteins. Separation ofreduced species from the reducing agent requires less effort and is moreefficient. Immobilized reducing agent can be regenerated as is known inthe art and reused multiple times. The immobilized dithioamines can beS-acylated (particularly S-acetylated) for chemical or enzymaticactivation prior to use. In specific embodiments, preferred S-acylateddithioamines are also N-acylated. For example, S-acylated dithioaminescan be deacylated employing hydroxyl amine or other such art-recognizeddeacylating reagents. The dithioamine reducing agents can be immobilizedon any appropriate surface by any immobilization method known in theart. In specific embodiments, dithioamine reducing agents can becovalently attached to a surface by reaction of a reactive group on thereducing agent with a reactive group on the surface. Alternatively, ahomo- or heterobifunctional crosslinking reagent such as are known inthe art can be employed to immobilize the dithioamine reducing agent ofthis invention on the surface.

S-acylated dithioamines function as precursors of the dithioaminereducing agents hereof which can be activated as reducing agents byremoval of the S-acyl groups to generate sulfhydryl groups. In morepreferred embodiments of S-acylated dithioamines, one of R₆ or R₇ is anacyl group to avoid substantial acyl transfer to the —NR₆R₇ group. Inother preferred embodiments, both of R₆ and R₇ are groups other thanhydrogen, e.g., one of R₆ or R₇ is an alkyl group and one or R₆ or R₇ isan —COR₁₃ group as defined above. S-acylated dithioamines of thisinvention can be activated by removal of acyl groups as is known in theart, for example by treatment with hydroxylamine or by treatment withacidic methanol as illustrated in Scheme 1. Of particular interest isactivation of S-acylated dithioamines with esterases, includingcarboxylesterases. Useful esterases that function for removal of S-acylgroups are known in the art, such as those esterases that are associatedwith the removal of SATE (S-acyl-2-thioethyl) protection [30, 31, 32].

In a specific embodiment, S-acylated precursors of reducing agentsherein are activated in vivo, e.g., inside of cells by the action ofesterases therein. For example, these precursors can be activated insideof mammalian cells by the action of mammalian esterase. Morespecifically, these precursors can be activated inside of human cells bythe action of human esterases. It is noted that such esterase may alsobe employed in vitro for activation of S-acylated precursors.

The invention provides dithioamine reducing agents conjugated to variousspecies T which can be biological molecules, such as proteins,carbohydrates or nucleic acids; labels or tags, such as radiolabels,isotopic labels or fluorescent labels or ligands or substrates thatselectively bind to target the conjugate to species which are to beselectively reduced. In specific embodiments, the reducing agents ofthis invention can be targeted for reduction of a specific proteinemploying such selective ligands or substrates. In specific embodiments,ligands can be mono- or disaccharides, e.g., glucose or fructose. Inspecific embodiments, ligands can be sialic acid or analogues thereof(see, exemplary sialic acid analogues in ref. 33). Reference 33 isincorporated by reference herein in its entirety for its description ofsialic acid analogues.

As noted above, dithioamine reducing agents of this invention optionallycarry -M or -L-T groups which function for immobilization, optionallyspacing, and/or conjugation to surfaces or other chemical or biologicalmoieties, i.e., T. In specific embodiments, -M is a reactive group or aspacer moiety or linker (-L-) carrying a reactive group wherein thereactive group reacts with one or more of: an amine group, a carboxylicacid group, a sulfhydryl group, a hydroxyl group, an aldehyde or ketonegroup, an azide group, an activated ester group, a thioester group, orphosphinothioester, or reacts with one reactive group of ahomobifunctional or a heterobifunctional crosslinking reagent. Inanother embodiment, M is or contains a reactive group that can beligated to a peptide or protein by a peptide ligation method. Inspecific embodiments, M is or contains an amine group, a carboxyl groupor ester thereof, an activated ester group, an azide, a thioester, or aphosphinothioester. In another embodiment, M is or contains a latentreactive group which can be selectively activated for reaction.

In general the optional spacer moiety of the M group is compatible withthe reactive group therein (e.g., does not detrimentally affectreactivity of the reactive group) and the spacer itself is not reactivewith the compounds to be conjugated or surfaces on which the reagent isto be immobilized. In specific embodiments, the spacer moiety containsfrom 3-20 atoms (typically C, O, S and/or N atoms which may besubstituted with H or non-hydrogen substituents), including residuesfrom the reactive group), and optionally contains one or morecarbon-carbon double bonds, and/or a 5- to 8-member alicyclic, a 5- to8-member heterocyclic, a 6- or 10-member aryl or a 5- or 6-memberheteroaryl ring. Carbon atoms in the spacer or linker are optionallysubstituted with one or more hydroxyl groups, oxo moieties (═O), orhalogens (e.g., F). Nitrogen groups in the spacer may be substitutedwith hydrogen and/or with C1-C3 alkyl groups. The spacer may contain adiol (>C(OH)—C(OH)<) moiety. The spacer may be selectively cleavable bychange of conditions (e.g., pH change), addition of a cleavage reagent,or photo irradiation (e.g., UV irradiation). In specific embodiments, acleavable spacer includes a diol moiety which is selectively cleavableby treatment for example with periodate, an ester moiety, which isselectively cleavable by treatment with hydroxylamine, or a sulfonemoiety (—SO₂—) which is selectively cleavable under alkaline conditions.

In specific embodiments, -M is selected from —X or -L-X, where X is thereactive group for ligation, bonding or crosslinking to an amino acid,peptide or protein and -L- is a divalent linker or spacer moiety.

A variety of spacer or linker moieties -L- are known in the art to beuseful for bioconjugation or immobilization. In specific embodiments,the linker may be a bond. All such art known spacer moieties can beemployed in this invention, if compatible with the chemistry of thedithioamine reagent and the species or surface to which the reagents isto be conjugated or upon which it is to be immobilized. The spacermoiety should not detrimentally affect reactivity of chosen reactivegroups and should not itself react with the dithioamine reagent, anyreactive group employed for conjugation or immobilization or with thesurface or species to be conjugated to the reagent.

In specific embodiments -L- is selected from the following divalentmoieties:

-   —Y1-L1-Y3-, where Y1 and Y3 are optional and may be the same or    different;-   —Y1-L1-L2-Y3-, where Y1 and Y3 are optional and may be the same or    different and L1 and L2 are different; or-   —Y1-L1-[L2-Y2]y-L3-Y3-, where Y1 and Y3 are optional, Y1, Y2 and Y3    may be the same or different, L1 and L3 are optional and L1, L2 and    L3 may be the same or different and y is an integer indicating the    number of repeats of the indicated moiety;

wherein each L1-L3 is independently selected from an optionallysubstituted divalent aliphatic, alicyclic, heterocyclic, aryl, orheteroaryl moiety having 1 to 30 atoms and each Y1, Y2 and Y3 isindependently selected from: —O—, —S—, —NRc-, —CO—, —O—CO—, —CO—O—,—CO—NRc-, —NRc-CO—, —NRc-CO—NRc-, —OCO—NRc-, —NRc-CO—O—, —N═N—,—N═N—NRc-, —CO—S—, —S—CO—, —SO₂—, —CRc(OH)—CRc(OH)—, where Rc ishydrogen or C1-C3 alkyl.

In specific embodiments, y is 1-12 and L1-L3 are selected from: —(CH₂)y-(an alkylene) wherein one or more, and preferably 1-4, carbons of thealkylene are optionally substituted with one or more non-hydrogensubstituents selected from halogens, C1-C3 alkyl groups or hydroxylgroups, preferred y are 2-6;

a cycloalkylene, having a 3-8-member ring wherein one or more, andpreferably 1-4, carbons of the cycloalkylene are optionally substitutedwith one or more non-hydrogen substituents selected from halogens, C1-C3alkyl groups or hydroxyl groups, including among others a1,4-cyclohexylene, a 1,3-cylohexylene, a 1,2-cyclohexylene; a1,3-cyclopentylene, each of which is optionally substituted;

a phenylene, wherein 1-4 of the ring carbons are optionally substitutedwith one or more non-hydrogen substituents selected from halogens, C1-C3alkyl groups, nitro group, cyano group, or hydroxyl groups, including a1,4-phenylene, a 1,3-phenylene or a 1,2-phenylene, each of which isoptionally substituted;

a naphthylene, wherein 1-8 of the ring carbons are optionallysubstituted with one or more non-hydrogen substituents selected fromhalogens, C1-C3 alkyl groups, nitro group, cyano group, or hydroxylgroups, including a 2,6-naphthylene, a 2,7-naphthylene, a1,5-naphthylene, or a 1,4-naphthylene moiety, each of which isoptionally substituted;

a biphenylene, wherein 1-8 of the ring carbons are optionallysubstituted with one or more non-hydrogen substituents selected fromhalogens, C1-C3 alkyl groups or hydroxyl groups, including a1,4′-biphenylene, a 1,3′-biphenylene or a 1,2′-biphenylene, each ofwhich is optionally substituted;

an alkenylene, i.e., a divalent alkylene group, containing one or more,preferably 1 or 2 double bonds and having 2-12 and preferably 2-8 carbonatoms, wherein one or more, and preferably 1-4, carbons are optionallysubstituted with one or more non-hydrogen substituents selected fromhalogens, C1-C3 alkyl groups or hydroxyl groups, including among others,—CH═CH— and —CH═CH—CH═CH— which are optionally substituted;

a heterocyclene (i.e., a divalent heterocyclic moiety) having a3-8-member ring with 1-3 heteroatoms, selected from N, O or S, whereinone or more, and preferably 1-4 carbons, or where feasible heteroatoms,of the heterocyclene are optionally substituted with one or morenon-hydrogen substituents selected from halogens, C1-C3 alkyl groups,nitro groups, or hydroxyl groups, including among others a 2,4-3H-azepinylene moiety, a piperidinylene (e.g., a 1,4-piperidinylene),a piperazinylene (e.g., a 1,4-piperazinylene), a triazolidinylene (adivalent triazolidinyl) or a triazolylene (a divalent triazolyl) each ofwhich is optionally substituted; or

a heteroarylene (i.e., a divalent heteroaryl moiety) having a 5- or6-member heteroaryl ring having 1-3 heteroatoms selected from N, O or S,wherein one or more, and preferably 1-2 carbons, or where feasibleheteroatoms, of the heteroarylene are optionally substituted with one ormore non-hydrogen substituents selected from halogens, C1-C3 alkylgroups, nitro groups, or hydroxyl groups, including among others apyridylene (e.g., 2,5-pyridylene), imidazolylene (e.g.,2,5-imidazolylene, 4,5-imidazolylene), each of which is optionallysubstituted.

In additional embodiments, the spacer is an ethylene glycol spacer. Morespecifically, -L- is selected from —[(CH₂)y-O]a-, where y is 1-4 and ais 1-6, and preferably 1-3.

In further embodiments, -M is selected from:

-   —CO—NH—CRaRb—[CO—NH—CRaRb]a-CO—OH, where a is 1-6;-   —COO—CRaRb—[CO—NH—CRaRb]a-CO—OH, where a is 1-6;-   —O—CO—NH—CRaRb—[NH—CO—CRaRb]a-NH₂, where a is 1-6;-   —Y4-CRaRb—[W—CRaRb]a-X4, where W is —NH—CO— or —CO—NH—, where a is    1-6;-   where:-   —X4 is a functional group that reacts with one or more of an amine    group, a carboxylic acid group or ester thereof, a sulfhydryl group,    a hydroxyl group, an azide group, a thioester group, a    phoshinothioester group, an aldehyde group or a ketone group of an    amino acid, peptide or protein; and-   —Y4- is —O—, —S—, —NH—, —CO—, —CO₂—, —O—CO—, —CO—O—, —CO—NRc-,    —NRcCO—, —CO—S—, or —S—CO— and Rc is hydrogen or a C1-C3 alkyl;-   Ra and Rb are selected independently from hydrogen, a C1-C8    aliphatic group, an alicylic, a heterocyclic, an aryl or a    heteroaryl group, each of which is optionally substituted or-   Ra is hydrogen and Rb is a side-group or protected side-group of a    proteinogenic amino acids or an amino acid selected from    hydroxyproline, ornithine, or citrulline.

In specific embodiments, X and X4 are —NH₂, —COON or an activated esterthereof, —SH, —N₃, —COH, —CO—CH═CH₂, —NH—CO—CH═CH, or —C≡CH.

In specific embodiments, the reducing agent of formula I, IA or IBexcept for the -M group or any salt counterion thereof contains at most20 carbon atoms.

In specific embodiments, in the reducing agent of formula I, IA or IB,none of R₁-R₅ or R₈ is an —N(R₉)₂ group where one or both of R₉ ishydrogen or an alkyl group. In specific embodiments, in the reducingagent of formula I, IA or IB, none of R₁-R₅ or R₈ carries an amine group—N(R₉)₂ group where one or both of R₉ is hydrogen or an alkyl group. Inspecific embodiments, in the reducing agent of formula I, IA or IB, noneof R₁-R₅ or R₈ is a hydroxyl group. In specific embodiments, in thereducing agent of formula I, IA or IB, none of R₁-R₅ or R₈ issubstituted with a hydroxyl group.

T is a biological or chemical species or a surface to which a reducingagent is conjugated, typically via a spacer or linking moiety (-L-). Tcan be a biological molecule, which includes molecules derived fromnature such as peptides, proteins, carbohydrates (e.g., mono-, di- andoligosaccharides), or nucleic acids (e.g., a nucleoside, a mono-, di- orpolynucleotide, a DNA sequence or an RNA sequence) which may be isolatedfrom nature or synthesized. T can be a biological or chemical specieswhich is a ligand which binds to a biological molecule or which is asubstrate for an enzyme. T can, for example, be a biological or chemicalspecies which is anionic or cationic which will preferentially associatewith a corresponding cationic or anionic portion, respectively, of abiological molecule, such as a peptide or protein to target thebiological molecule and selectively target the reducing agent (oracylated precursor thereof) of this invention to the biological moleculeto affect its biological activity.

In a particular embodiment, T is a pharmacophore of a selectedbiologically active species, particularly a pharmacophore associatedwith a selected peptide or protein or ligands thereof. As is known inthe art, pharmacophore refers to the 3-D molecular features (structuraland electronic features) necessary for interaction with a targetbiological species which can trigger or block a biological response.Pharmacophore modeling [29] represents one aspect of ligand-based drugdesign which can provide 3-D chemical moieties which interact withbiological molecules (e.g., by binding or association there with) andthereby affect biological function thereof.

In specific embodiments, dithioamines of this invention can beimmobilized on various surfaces including inorganic and organicsurfaces. The surface may be among others that of a plate, a container(tube, bottle, etc.) which can, for example, be made of plastic orglass, a bead, particle, microparticle or nanoparticle. The surface maybe a polymer, a co-polymer, a block co-polymer, a graft-copolymer or aresin each of which may be cross-linked. Polymeric materials includeamong others, agarose, poly(acrylamide) and co-polymers thereof,poly(methylmethacrylate) and co-polymers thereof, poly(hydroxyethylmethacrylate) and co-polymers thereof, poly(vinyltoluene) andco-polymers thereof, poly(styrene) and co-polymers thereof (e.g.,poly(styrene/divinylbenzene) copolymers, poly(styrene/acrylate)co-polymers, poly(styrene/butadiene) co-polymers,poly(styrene/vinyltoluene) co-polymers). The surface may be that of acore-shell particle having a core of one material (e.g., one polymericmaterial) and a shell or coating of a different material (e.g., acoating polymers, such as poly(ethylene glycol), poly(vinyl alcohol),poly(acrylamide), poly(vinyl pyrrolidone), among others. A specificcore-shell particle has a poly(styrene) core with a poly(hydroxyethylmethacrylate) shell.

The surface may be glass, quartz, silica, silica gel, alumina or othermetal oxides, or inert metal such as gold (e.g., gold nanoparticles),again in the form among others of plates, beads or particles. Thesurface may be that of a magnetic, paramagnetic or superparamagneticmaterial.

The surfaces may contain reactive functional groups that derive from thematerial from which the particle is made (e.g., OH groups on glass, oramine groups of poly(acrylamide)) or the particles may be functionalizedas is known in the art with reactive groups, for example, as notedabove, including among others, amine groups, aldehyde or ketone groups,carboxyl groups, epoxides, hydrazides, hydroxyl, amide, sulfamyl groups,or activated esters, such as tosyl-, mesyl- or tresyl-activated estersor NHS esters.) Hermanson, G. T. (2008) Bioconjugate Techniques (SecondEdition) Academic Press, N.Y., Chapter 14, pages 582-625 describedconjugation/immobilization of various chemical and biological species onsurfaces and particles. This reference is incorporated by referenceherein in its entirety for this description.

In a specific embodiment, one or more reducing agents of this inventionare conjugated to a polymer (i.e., T is a polymer). In a specificembodiment, two or more molecules of reducing agent are conjugated to apolymer. In a specific embodiment, 10% or more or 25% or more of themonomer groups of a polymer are conjugated to a reducing agent offormulas I, IA or IB. In specific embodiments, the polymer carries oneor more amino groups which can be conjugated, optionally but preferablyvia a spacer or linker, to a reducing agent or acylated precursorthereof of this invention. In specific embodiments, the polymer carriesone or more amine, amide or ester side chains which can be conjugated toone or more reducing agents of this invention. Polymers useful for suchconjugation include among many others, poly(lysine), poly(ornithine),poly(lysine ornithine), poly(aspartic acid), poly(glutamic acid),poly(acrylamide), poly(ethylene imine), poly(propylene imine),poly(allyl amine), poly(vinyl amine), poly(2-aminoethyl methacrylate),poly(methacrylate), poly(methyl methacrylate), poly(acrylate),poly(hydroxyethyl methacrylate), poly(methyl acrylate), poly(vinylacetate) and copolymers including block copolymers thereof. Methods ofconjugation as described in Hermanson, G. T. (2008) BioconjugateTechniques (Second Edition) Academic Press, N.Y. can be employed orreadily adapted for conjugation to polymers.

In specific embodiments, the dithioamine reducing agents or dithianeprecursors thereof are covalently attached to surfaces, in particular toresins, which may be in the form of beads or other particles or in theform of coatings on surfaces or particles. In specific embodiments,resins useful for immobilization of dithioamines or dithianes of thisinvention include resins known and used in the art for solid-phaseorganic synthesis and/or for solid-phase peptide synthesis. A variety ofsuch resins are known in the art and are commercially available or canbe prepared by methods that are well-known in the art. For example, suchresins are or may be functionalized with amine, azide, carboxyl,sulfamyl, formyl, halogen, hydroxyl, mercapto, sulfonylchloride,sulfonic acid, or various activated ester groups for reaction withappropriate reactive groups attached to the dithioamine or dithiane toimmobilize the dithioamine or dithiane. Resins are typically composed ofa polymer matrix which may be cross-linked, such as polystyrene, andfunctional groups may be directly attached to the resin or attached to alinker groups, such as polyethylene glycol, which are in turn attachedto the resin matrix. In some cases, resins may contain latent reactivegroups (e.g., protected reactive groups) which must be activated beforethe dithioamine or dithiane is immobilized. Useful resins forimmobilization of dithioamines or dithianes of this invention includeamong others, aminoalkyl resins, Rink amide resin, MBHA resins, indoleresins, hydroxylamine resins, Sieber amide resins, PAL resins,sulfamyl-based resins, Wang resin, HMPA-AM resins, Merrifield resin, PAMresins, oxime resins, various safety-catch resins and the like. One ofordinary skill in the art can employ any such resins to immobilize thedithioamines and dithianes of this invention using well-known methods orroutine adaptation of such well-known methods. Albericio, F. andTulla-Puche (ed) (2008) The Power of Functional Resins in OrganicSynthesis (Wiley-Verlag) provided a description of various useful resinsand methods of using such resins for immobilization of various chemicalspecies. This reference is incorporated by reference herein in itsentirety to illustrate what is known in the art concerning such resins,methods for their use and methods of immobilization that are useful inthis invention.

The invention provides specific immobilized reducing agents andimmobilized dithianes which are precursors thereof of formulas:

where R is a surface including a resin,

-   L is a divalent linker as described herein,-   R₂-R₆ are as defined above, and-   X1 is a bond, —O—, —OCO—, —COO—, —NHCO—, —CONH—, —SO₂—NH—,    —SO₂—NH—CO—, —NHCONH—, or —OCOO—.

In specific embodiments, R₂-R₆ are all hydrogens. In specificembodiments, —X1-L- is —NHCO—(CH₂)r-, where r is 1-6 and r is preferably2; or —X1-L- is —NHCO—CH₂CH₂OCH₂CH₂—.

In specific embodiments, compounds of this invention of formulas I, IA,IB and II contain a reactive functional group for attachment of thecompound to a T species (as described herein, a polymer or a surface.The reactive functional group can, for example, be a group that reactswith an amine, a carbonyl, a carboxylate, a carboxylic ester, sulfamylgroup (—SO₂—NH₂), or hydroxyl group. Generally, sulfhydryl reactivegroups are less preferred as care must be taken to protect sulfhydrylgroups on the reducing agent. Preferably such reactive groups react toconjugate the species under conditions such that the reducing agentsubstantially retains functionality and which do not substantiallydetrimentally affect biological activity of interest of the T species(if any). A variety of reactive groups useful for such coupling areknown in the art and one of ordinary skill in the art can select amongsuch known reactive groups to practice the methods of the presentinvention without undue experimentation.

An overview of bioconjugation methods that can be employed in thepresent invention is found in Hermanson, G. T. Bioconjugation Techniques(2nd Ed.) 2008 Academic Press/Elsevier London, UK. This reference alsocontains detailed descriptions of homobifunctional andheterobifunctional crossing linking reagents which can be employed forconjugation.

Amine-reactive groups are exemplified by a carboxylate group, acarboxylate ester group, an acid chloride group, an aldehyde group, anacyl azide group, an epoxide, an isothiocyanate group, an isocyanategroup, an imidoester group or an anhydride group. Amines react withcarboxylates in the presence of coupling reagents, such ascarbodiimides. Amine-reactive groups include active carboxylic acidester groups, such as succinimidyl ester groups or sulfosuccinimidylester groups (e.g., N—OH succinimidyl or N—OH sulfosuccinimidyl groups);haloalkyl ester groups, such as trifluoroalkyl ester groups andhexafluoroalkyl ester groups; halophenyl ester groups, particularlyfluorophenyl and chlorophenyl ester groups, including penta- andtetrafluorophenyl ester groups, pentachlorophenyl ester groups;nitrophenyl ester groups, including 2-nitrophenyl, 4-nitrophenyl and2,4-dinitrophenyl ester groups; as well as other substituted phenylester groups, including sulfodichlorophenol ester groups.

General conditions for carrying out reactions between amine-reactivegroups and amino groups of an amino acid, peptide or protein are wellknown in the art and can be carried out by one of ordinary skill in theart without undue experimentation.

Although not preferred, sulfhydryl-reactive groups are exemplified byhaloacetyl and haloacetamidyl groups, particularly iodoacetyl andbromoacetyl or corresponding acetamidyl groups, maleimide groups,haloalkyl groups, halobenzyl groups, acryloyl groups, epoxide groups,groups that undergo thiol-disulfide exchange, such as dipyridyldisulfide groups or 2,2′-dihydroxy-6,6′-dinaphthyldisulfide groups, orthiosulfate groups. General conditions for carrying out reactionsbetween sulfhydryl-reactive groups and sulfhydryl groups are well knownin the art and can be carried out by one of ordinary skill in the artwithout undue experimentation.

Carboxylate-reactive functional groups are exemplified by amines (e.g.,employing a carbodiimide), hydrazine groups, hydrazide groups,sulfonylhydrazide groups, diazoalkyl groups, sulfamyl, diazoaryl groups,diazoacetyl groups, hydroxyl groups or sulfhydryl groups.

Hydroxyl-reactive functional groups are exemplified by isocyanategroups; epoxide groups; alkyl or aryl halide group, e.g., a halotritylgroup; an activated carbamate group, an activated ester group (such asdescribed above), N,N′-disuccinimidyl carbonate groups orN-hydroxysuccinimidyl chloroformate groups. Aldehyde and ketone-reactivegroups are exemplified by hydrazine groups and derivatives thereofincluding hydrazides, semicarbazides and carbohydrazides, and aminogroups. Various methods for introduction of aldehyde and ketone groupsinto amino acids, peptides and proteins are known in the art.

Azide groups react with alkenyl or akynyl groups (in so-called Clickreactions) to form triazolines or triazoles. Click reactions can be usedto link a reducing agent of the invention with a T group or to asurface. Linkers formed in such reactions will include a triazoline ortriazole moiety.

Phosphinothioesters react with azide groups as described in U.S. Pat.Nos. 6,972,320 and 7,256,259, and 7,317,129 and U.S. publishedapplication US 2010/0048866 to form amide bonds in a tracelessStaudinger ligation. Phosphinothioesters can be prepared employingphosphinothiol reagents as also described in these references. Each ofthese references is incorporated by reference herein in its entirety fordescriptions of such ligation reactions, methods of making azides andmethods of making phosphinothioesters.

Aldehyde, ketone, azide activated esters groups, thioester,phosphinothiol groups are introduced by any art-known methods.

Homobifunctional crosslinking reagents contain two identical reactivegroups separated by a spacer or linker moiety. Heterobifunctionalcrosslinking reagents contain two reactive groups with differentselectivity for reaction, e.g., an amine-reactive group and asulfhydryl-reactive group separated by a spacer or linker moiety.Various homobifunctional and heterobifunctional crossing linkagereagents are known in the art and a number are commercially availablefrom Pierce (Thermo Scientific), Rockford, Ill., Sigma-Aldrich, St.Louis, Mo. or Molecular Probes (Life Technologies), Eugene Oreg.

Useful homobifunctional crosslinking reagents include those carrying twoamine-reactive groups, those carrying two carboxylate reactive groups,or those carrying two aldehyde or ketone reactive groups.Homobifunctional crosslinking reagents carrying sulfhydryl reactivegroups are generally not preferred. Such reagents can be employed ifappropriate sulfhydryl group protecting agents, such as acyl groups, areemployed to prevent reaction with the sulfhydryl groups of the reducingagent.

Useful heterobifunctional crosslinking reagents include those carryingone of an amine-reactive group, a sulfhydryl reactive group, acarboxylate reactive group, or an aldehyde or ketone reactive group andone of a different reactive group selected from an amine-reactive group,a sulfhydryl reactive group, a carboxylate reactive group, or analdehyde or ketone reactive group. Again heterobifunctional crosslinkingreagents carrying sulfhydryl reactive groups are generally not preferredas noted above.

Homobifunctional and heterobifunctional crosslinking reagents can ingeneral contain any spacer or linking moiety compatible with thereactive groups therein wherein the spacer or linker itself is notreactive with the compounds to be conjugated. In specific embodiments,disulfide moieties are not preferred in such spacers or linkers. Inspecific embodiments, the spacer or linking moiety typically ranges from3-20 atoms (typically C, O, S and/or N atoms) in length (includingresidues from the reactive group), and optionally contain one or morecarbon-carbon double bonds, and/or a 5- or 6-member alicyclic,heterocyclic, aryl or heteroaryl ring. Carbon atoms in the spacer orlinker are often substituted with one or more hydroxyl groups, oxomoieties (═O), or halogens (e.g., F). Nitrogen groups in the linker maybe substituted hydrogen or with C1-C3 alkyl groups. The spacer or linkermay contain one or two —SO₂— moieties. The spacer or linker may beselectively cleavable by change of conditions (e.g., pH change),addition of a cleavage reagent, or photoirradiation (e.g., UVirradiation). In specific embodiments, a cleavable linker includes acleavable linker contains a diol moiety which is selectively cleavableby treatment for example with periodate, an ester moiety, which isselectively cleavable by treatment with hydroxylamine, a sulfone moiety(—SO₂—) which is selectively cleavable under alkaline conditions.

Homobifunctional crosslinking reagents can be used, for example, toconjugate an amine group of an reducing agent or acylated precursorthereof with an amine functionality on a biological or chemical speciesT, polymer or a surface. Amine-reactive groups employed inhomobifunctional crosslinking reagents include among others, activatedester groups, such as NHS esters (N-hydroxysuccinimide esters) or sulfoNHS esters (N-hydroxysulfosuccinimide esters), imidoester group, such asmethylimidate salts, isothiocyanate groups and aryl halide groups, suchas difluorobenzene derivatives. Amine-reactive homobifunctional includeamong others: dithiobis(succinimidylproprionate) [DSP] and its sulfo-NHSanalog [DTSSP], disuccinimidyl suberate [DSS] and its sulfo-NHS analog[BS3], disuccinimidyl tartarate [DST] and its sulfo NHS analog[sulfo-DST], bis(2-succinimidyloxy-carbonyloxy)ethylsulfone [BSOCOES]and its sulfo-NHS analog [sulfo-BSOCOES], ethylene glycolbis(succinimidylsuccinate) [EGS] and its sulfo-NHS analog [sulfo-EGS],disuccinimidyl glutarate [DSG], N,N′-disuccinimidyl carbonate [DSC],dimethyl adipimidate [DMA], dimethyl 3,3-dithiobispropionimidate [DTBP],4,4′-disiothiocyanatostilbene-2,2′-disulfonic acid salts,1,5-difluoro-2,4-dinitrobenzene [DFDNB],4,4′-difluoro-3,3′-dinitrodiphenylsulfone.

Hydroxyl-reactive homobifunctional crosslinking reagents can be used toconjugate a hydroxyl group on a reducing agent or acylated precursorthereof with a hydroxyl group substituent on a chemical or biological Tspecies, a polymer or a surface. Hydroxyl-reactive groups include thosehaving epoxide groups, such as diglycidylethers, particularly1,4-butanediol diglycidyl ether.

A carboxylate group on a reducing agent or acylated precursor thereofcan be conjugated to a carboxylate group substituent on a chemical orbiological T species, a polymer or a surface, for example, by generatingan active ester at the carboxylate groups and esterifying the activeesters with an alkanediol crosslinking reagent, such a 1,6-hexane diol,or 1,12-dodecanediol.

Aldehyde/ketone-reactive homobifunctional crosslinking reagents can beused to conjugate an aldehyde or ketone group of a reducing agent oracylated precursor thereof of this invention with an aldehyde or ketonegroup substituent on a phenylboronate compound. Bis-hydrazide reagentscan be used to crosslink molecules containing aldehyde or ketone groups;examples of such crosslinking reagents include among others adipic aciddihydrazide and carbohydrazide.

Heterobifunctional crosslinking reagents include those which contain anamine reactive group and a sulfhydryl-reactive group. For example, sucha heterobifunctional crosslinking reagent can be used to link an aminegroup on a compound of this invention with a sulfhydryl substituent in aT species of this invention.

Exemplary heterobifunctional crosslinking reagents include thosecarrying an activated ester group, such as an NHS ester (or sulfo-NHSester) group or a nitrophenyl or other substituted phenyl ester and amaleimide group; those carrying such an activated ester group and adithiopyridyl group, those carrying an activated ester group and anhaloacetyl group (e.g., an iodoacetyl group), or those carrying animidoester group and a maleimide group.

Exemplary heterobifunctional amine/sulfhydryl-reactive crosslinkingreagents include, among others, N-(γ-maleimidobutyryloxy)succinimideester [GMBS] and its sulfo-NHS analog [sulfo-GMBS],4-succinimidyloxycarbonyl-α-(2-pyridyldithio)toluene [SMPT],succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate [SMCC] andits sulfo-NHS analog [sulfo-SMCC],m-maleimidobenzoyl-N-hydroxy-succinimide ester [MBS] and its sulfo-HNSanalog [sulfo-MBS], N-succinimidyl(4-iodoacetyl)-aminobenzoate [SIAB]and its sulfo-HNS analog [sulfo-SIAB],succinimidyl-6-(iodoacetyl)aminohexanoate [SIAX],N-succinimidyl-3-92-pyridylthio)propionate [SPDP],succinimidyl-4-(p-maleimidophenyl)butyrate [SMPB] and its sulfo-NHSanalog [sulfo-SMPB],succinimidyl-([N-maleimidopropionamidol]ethyleneglycol esters [SM(PEG)n,where n is 4, 6, 8, 12, 24] and p-nitrophenyl iodoacetate [NPIA],N-hydroxysuccinimidyl 2,3-dibromopropionate [SDBP].

Heterobifunctional crosslinking reagents also include those whichcontain one of an amine-reactive, carboxylate-reactive orcarbonyl-reactive group and a photoreactive group which is activated onirradiation to reactive with various reactive groups, includingnucleophiles, reactive hydrogen, active hydrogen amines or olefins.

It will be appreciated that it may be necessary dependent upon theconjugation method employed to acylate or otherwise protect thesulfhydryl groups of the reducing agent or other potentially reactivegroups therein from undesired conjugation. Useful thiol protectivegroups, amine protecting groups and protective groups for various otherreactive groups are known in the art, for example as described in Wutts,P. G. and Greene, T. (2007) Green's Protecting Groups in OrganicSynthesis (Fourth Edition) John Wiley & Sons, N.Y.

The compounds of formulas I, IA, IB and II can be in the form of salts,for example ammonium (—NR₆R₇H⁺) salts, with a selected anion orquaternized ammonium salts (e.g., —NR₆R₇R₂₀ ⁺, where R₂₀ is a C1-C3alkyl group). The salts can be formed as is known in the art by additionof an acid to the free base. Salts can be formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, or organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid,N-acetylcystein and the like.

In specific embodiments, compounds of the invention can contain one ormore negatively charged groups (free acids) which may be in the form ofsalts. Exemplary salts of free acids are formed with inorganic baseinclude, but are not limited to, alkali metal salts (e.g., Li⁺, Na⁺,K⁺), alkaline earth metal salts (e.g., Ca²⁺, Mg²⁺), non-toxic heavymetal salts and ammonium (NH₄ ⁺) and substituted ammonium (N(R′)4+salts, where R′ is hydrogen, alkyl, or substituted alkyl, i.e.,including, methyl, ethyl, or hydroxyethyl, specifically, tri methylammonium, triethyl ammonium, and triethanol ammonium salts), salts ofcationic forms of lysine, arginine, N-ethylpiperidine, piperidine, andthe like. Compounds of the invention can also be present in the form ofzwitterions. Compound of formulas I, IA, IB and II also include thosewhich are pharmaceutically acceptable salts, which refers to those saltswhich retain the biological effectiveness and properties of the freebases or free acids, and which are not biologically or otherwiseundesirable.

The scope of the invention as described and claimed encompasses theracemic forms of the compounds as well as the individual enantiomers andnon-racemic mixtures thereof. The compounds of the invention may containone or more asymmetric carbon atoms, so that the compounds can exist indifferent stereoisomeric forms. The compounds can be, for example,racemates or optically active forms. The optically active forms can beobtained by resolution of the racemates or by asymmetric synthesis. In apreferred embodiment of the invention, enantiomers of the inventionexhibit specific rotation that is + (positive). Preferably, the (+)enantiomers are substantially free of the corresponding (−) enantiomer.Thus, an enantiomer substantially free of the corresponding enantiomerrefers to a compound which is isolated or separated via separationtechniques or prepared free of the corresponding enantiomer.“Substantially free,” means that the compound is made up of asignificantly greater proportion of one enantiomer. In preferredembodiments the compound is made up of at least about 90% by weight of apreferred enantiomer. In other embodiments of the invention, thecompound is made up of at least about 99% by weight of a preferredenantiomer. Preferred enantiomers may be isolated from racemic mixturesby any method known to those skilled in the art, including highperformance liquid chromatography (HPLC) and the formation andcrystallization of chiral salts or prepared by methods described herein.See, for example, Jacques, et al., Enantiomers, Racemates andResolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al.,Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of CarbonCompounds (McGraw-Hill, N.Y., 1962); Wilen, S. H. Tables of ResolvingAgents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of NotreDame Press, Notre Dame, Ind. 1972).

The invention provides methods for reducing or preventing disulfide bondformation in one or more molecules having one or more sulfhydryl groupswhich comprise the step of contacting the one or more molecules with oneor more compounds of this invention, particularly one or more compoundsof formulas I, IA or IB. In a specific embodiment, the methods arecarried out under physiological conditions. In a specific embodiment,the methods are carried out at a pH ranging from 6-8. In a specificembodiment, the methods are carried out at a pH ranging from 6.5 to 7.5.In a specific embodiment, the methods are carried out employing one ormore compounds of the invention which are covalently attached to asurface. In specific embodiments, the surface is organic or inorganicwith specific examples of such surfaces provided herein. In a specificembodiment, the one or more compounds are S-acylated and the acyl groupsare removed to activate the one or more compounds as reducing agents.Activation of the S-acylated compounds can occur prior to contacting theone or more molecules carrying sulfhydryl groups. Activation of theS-acylated compounds can occur at about the same time as contacting theone or more molecules carrying sulfhydryl groups. For example, thecontacting step and S-acyl group removal can occur in tissue or in acell which contains one or more esterases which function for removal ofthe acyl groups.

In a specific embodiment, reducing or preventing disulfide bondformation reduces or prevents the formation of dimers or other oligomersof the one or more molecules having sulfhydryl groups. In a specificembodiment, reducing or preventing disulfide bond formation functions tomodulate a biological activity of the one or more molecules havingsulfhydryl groups. Modulation of the biological activity includes areduction in such activity or an enhancement of such activity. Inspecific embodiments, the one or more molecules carrying sulfhydrylgroups are biological molecules, more specifically are biologicalmacromolecules and yet more specifically are peptides, proteins,carbohydrates or nucleic acids.

In a specific embodiment, the molecules having one or more sulfhydrylgroups are peptides or proteins and reducing or preventing disulfidebond formation functions to modulate a biological activity of the one ormore peptides or proteins. In a specific embodiment, reducing orpreventing disulfide bond formation functions to reduce a biologicalactivity of a peptide or protein. In another embodiment, reducing orpreventing disulfide bond formation functions to enhance a biologicalactivity of a peptide or protein. In a specific embodiment, the peptideor protein is redox-sensitive peptide or protein, for example a peptideor proteins the biological activity of which is affected by oxidativestress as is described in Cumming et al. [34]. Cumming et al. isincorporated by reference herein for its description of suchredox-sensitive peptides and proteins and specific examples giventherein.

Of particular interest for therapeutic application of the compounds ofthis invention are redox-sensitive peptides and proteins whose functionis associated with human or animal disease. Non-limiting specificexamples of such redox-sensitive peptides or proteins include PTEN(human phosphatase PTEN), SOD (superoxide dismutase), and pMK2 (anisoform of pyruvate kinase). Decreased PTEN activity is associated withmany cancers (i.e., PTEN activity is associated with cancer protection).A cysteine residue near the active site of human phosphatase PTEN isknown to be sensitive to oxidation, such that its activity is decreases.Prevention of this inactivation employing a reducing agent or precursorthereof of this invention can be of therapeutic benefit. Superoxidedismutase (Cu/Zn SOD) can be inactivated by the formation ofdisulfide-linked dimers. Decreased SOD activity is believed to be acause of amylotropic lateral sclerosis (ALS) (35, 36). Prevention ofdecreased SOD activity employing a reducing agent or precursor thereofof this invention can be of therapeutic benefit.

In specific embodiments herein, the dithioamine reducing agent, dithianeprecursor or acylated precursor thereof is conjugated to a biological orchemical species which targets or directs the conjugated reducing agentto a specific redox-sensitive peptide or protein. The biological orchemical species is for example a ligand, a substrate or a pharmacophoreof the target peptide or protein (which may be an enzyme).

PTEN is believed to function by attack/removal of a phosphoryl groupfrom C3 of the inositide below by a cysteine residue. This inositide isthe product of PTEN catalysis:

The PTEN cysteine can form a disulfide bond with another cysteineresidue inactivating PTEN. This inositide represents a pharmacophore ofPTEN and is highly anionic. To target a reducing agent or acylatedprecursor thereof to PTEN, a highly anionic chemical species can be usedwhich provides a substantial pharmacological equivalent of thephosphoinositol moiety.

A specific example of such a highly anionic chemical species is1,3,5-tricarboxybenzene. In an exemplary embodiment hereof one or morereducing agents, dithianes or acylated precursors thereof are conjugatedto T which is 1,3,5-tricarboxybenzene (or a salt thereof), via one ofthe carboxyl groups therein:

In another exemplary embodiment hereof one or more reducing agents oracylated precursors thereof are conjugated to T which is a1,3,5-tricarboxybenzyl group (or a salt thereof), via a spacer or linkergroup:

Conjugates of tricarboxybenzene with one or more of the reducing agentsof formulas I, IA or IB can be prepared by methods that are well-knownin the art, for example by methods that are described in Hermanson, G.T. (2008) Bioconjugate Techniques (Second Edition) Academic Press, N.Y.,for example Part I, Chapters 1 and 2. More specifically, a suitablereactive group can be installed on the 1,3,5-tricarboxybenzene and thefunctionalized T group can then be conjugated to a reducing agent oracylated precursor herein which carries an appropriate reactive group(as described herein above) employing an art-known homo- orheterobifunctional cross-linking reagent as described for example inHermanson, G. T. (2008) Bioconjugate Techniques (Second Edition)Academic Press, N.Y., Chapters 4 and 5.

More generally the invention provides dithioamine reducing agents,dithiane precursors or S-acyl precursors thereof which are targeted to acationic site for example which contain a pharmacophore of thephosphoinositol moiety. The invention provides compounds of formulas Vand VI:

and salts thereof,

-   where R₂-R₆ are as defined for formula I above,-   L is an optional divalent linker as defined above,-   A1 is an n-valent di- or tricarboxylic acid species which is    selected from an aliphatic group, a heterocyclic group, an aryl    group, or a heteroaryl group;-   R₂₀ is hydrogen, an alkyl group having 1-3 carbon atoms, an aryl    group, an arylalkyl group, wherein the alkyl, aryl or aryalkyl group    is optionally substituted with one or more halogens, and-   X1 is a bond, —O—, —CO—, —OCO—, —NHCO—, —COO—, or —CO—NH—.

In specific embodiments, A1 is a cycloalkyl or cycloalkenyl group having4-10 carbon atoms. In specific embodiments, A1 is an alkyl group having5-8 carbon atoms, a phenyl group, a benzyl group, a cyclohexyl group, acyclohexenyl group, a cyclopentyl group, a cyclobutyl group, a furan, atetrahydrofuran or a tetrahydropyran. In specific embodiments, the Algroup carries three carboxylic acid groups. In specific embodiments, X1is a bond. In specific embodiments L is —(CH₂)_(q)—, where q is 0, 1, 2,3, 4, 5, or 6. In specific embodiments, R₂-R₆ are all hydrogens. Inspecific embodiments, any R₂₀ are hydrogens or alkyl groups having 1-3carbon atoms.

In specific embodiments, A1 is a tricarboxylic acid species selectedfrom:

The carboxylic acid substituted A1 group may be a mixture ofregioisomers.

Reducing agents and acylated precursors thereof of formulas I, IA, andIB are useful as research reagents for reducing disulfide bonds andother redox applications, particularly in applications directed tobiological molecules, such as peptide, proteins, carbohydrates andnucleic acids. In various applications, reducing agents of thisinvention can be added to biological buffers. Compounds of formulas IIrepresent the oxidized form of the reducing agents of formulas I, IA andIB (noting that the specific chiral forms corresponding to formulas IAand IB are not specifically shown). These oxidized forms are also usefulas research reagents, for example, a combination of reduced and oxidizedcompounds of formulas I and II in appropriate ratio in solution (e.g.,aqueous solution) can provide a redox buffer with a selected reductionpotential. Redox buffers can be employed for example for refolding ofproteins.

One or ordinary skill in the art will recognize additional applicationsfor the reducing agents and acylated precursors thereof. For example,reducing agents are employed for treating hair, e.g. for removal of haircoloring and the like.

The term kit refers to kits including one or more of the reducingagents, or acylated precursors thereof or dithianes precursors thereofwhich are useful for preventing or inhibiting the formation of disulfidebonds or for cleaving disulfide bonds. In one embodiment, kits of thisinvention include one or more of the compounds of the present inventionor mixtures thereof and optionally reagents for ligating, or conjugatingsuch compounds with a biological or chemical species as discussedherein. The kits optionally include one or more solvents or buffers forapplication of a reducing agent of this invention. In anotherembodiment, kits of this invention include one or more compounds of thisinvention of this invention and optionally reagents, such as one or morehomo- or heterobifunctional crosslinking reagents, for ligating orconjugating to a surface. The kit may also include one or more surfaces,for example, in the form of plates, sheets, beads, particles,microspheres, microparticles, nanoparticles or the like to which acompound of this invention is to be immobilized or conjugated. A kit mayalso include a reagent for removing S-acyl groups of S-acyl precursorsof the reducing agents herein, such as hydroxylamine or an esterase.

Kits of the invention may comprise a carrier being compartmentalized toreceive in close confinement one or more containers, such as vials, testtubes, ampules, bottles and the like. Each of such container meanscomprises components or a mixture of components as described above(reducing agents, precursors, solvents or buffers, other reagents, etc.)The kits of the invention may further comprise one or more additionalcomponents (e.g., reagents and/or compounds) necessary or desirable forcarrying out one or more particular applications of the compositions ofthe present invention. In general kits may also contain one or morebuffers, control samples, carriers or recipients, vessels for carryingout one or more reactions, one or more additional compositions of theinvention, one or more sets of instructions, and the like. IN specificembodiments of kits herein the reducing agent is DTBA or a salt thereof.

The invention is also directed to art-known kits in which DTT therein isreplaced with one or more reducing agents of this invention andparticularly with DTBA or a salt thereof. Such its include DNA ligationor DNA blunting kits where DTT in buffers therein is replaced with oneor more reducing agents of this invention, particularly DTBA or a saltthereof. Kits of this invention also include kits for proteinpurification or protein assay kits which are compatible with reducingagents.

An aliphatic group as used herein refers to a monovalent non-aromatichydrocarbon group which include straight chain, branched, or cyclichydrocarbon groups which can be saturated or unsaturated with one ormore double bonds or one or more triple bonds. Aliphatic groups maycontain portions which are straight-chain or branched in combinationwith one or more carbon rings. Carbon rings of aliphatic groups maycontain one or more double bonds or one or more triple bonds. Carbonrings of aliphatic groups can contain 3- to 10-membered rings. Suchcarbon rings may be fused and may be bicyclic or tricyclic. Aliphaticgroups are optionally substituted with one or more non-hydrogensubstituents where optional substituents are described herein. Unlessotherwise specified, an aliphatic group can contain 1-20 carbon atoms orcan contain 1-10 carbon atoms. Aliphatic groups include those containing1-3, 1-6, and 1-8 carbon atoms. Aliphatic groups include, among others,alicyclic groups, alkyl groups, alkenyl groups and alkynyl groups.

An alicylic group as used herein refers to a monovalent non-aromaticcyclic hydrocarbon group which can be saturated or unsaturated with oneor more double bonds or one or more triple bonds. Alicyclic ringsinclude those containing 3- to 10-membered carbon rings. Alicyclicgroups include those containing one, two, three or more rings which maybe fused or linked by straight chain or branched alkylene, alkenylene oralkynylene moieties. Alicyclic groups include bicyclic and tricyclicrings. Alicyclic groups include those in which one or more carbon ringsare substituted with a straight-chain or branched alkyl, alkenyl oralkynyl group. To satisfy valence requirements, a ring atom may besubstituted with hydrogen or optionally with non-hydrogen substituentsas described herein. One or more carbons in an alicyclic group can be—CO— groups, i.e. a carbon can be substituted with an oxo (═O) moiety.Alicyclic groups are optionally substituted with one or morenon-hydrogen substituents where optional substituents are describedherein. Unless otherwise specified, an alicyclic group can contain 3-20carbon atoms or can contain 3-12 carbon atoms. Alicyclic groups includethose containing 3-6 and 3-8 carbon atoms. Alicyclic groups includeamong others cycloalkyl, cycloalkenyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl andcyclohexadienyl groups, all of which are optionally substituted.

A heterocyclic group as used herein refers to a monovalent non-aromaticcyclic hydrocarbon group wherein one or more of the rings contain one ormore heteroatoms (e.g., N, S, O, or P) which rings can be saturated orunsaturated with one or more double bonds or one or more triple bonds.In specific embodiments of this invention, heterocyclic rings which aresubstituents of the compounds of formulas IA and IB do not contain boronatoms. Heterocyclic rings include those containing 3- to 10-memberedrings where 1, 2 or 3 of the ring members are heteroatoms. Heterocyclicgroups include those containing one, two, three or more rings which maybe fused or linked by straight chain or branched alkylene, alkenylene oralkynylene moieties. Heterocyclic groups include bicyclic and tricyclicgroups. Heterocyclic groups include those in which a heterocyclic ringis substituted with a straight-chain or branched alkyl, alkenyl oralkynyl group. To satisfy valence requirements, a ring atom may besubstituted with hydrogen or optionally with non-hydrogen substituentsas described herein. One or more carbons in a heterocyclic group can be—CO— groups. One or more carbons in a heterocyclic ring can be—CO-groups. Heterocyclic groups are optionally substituted with one ormore non-hydrogen substituents where optional substituents are describedherein. Ring carbons and, where chemically feasible, ring heteroatomsare optionally substituted. Unless otherwise specified, a heterocyclicgroup can contain 3-20 carbon atoms, can contain 3-12 carbon atoms orcan contain 3-6 carbon atoms. Heterocyclic groups include thosecontaining one or two 4-, 5- or 6-member rings at least one of which hasone, two or three N, O or S atoms and wherein a ring optionally has oneor two double bonds. Heterocyclic groups include those containing asingle 5- or 6-member ring having one, two or three N, O or S atoms andoptionally having one or two double bonds. Heterocyclic groups includethose having 5- and 6-member rings with one or two nitrogens and one ortwo double bonds. Heterocyclic groups include those having 5- and6-member rings with an oxygen or a sulfur and one or two double bonds.Heterocyclic groups include those having 5- or 6-member rings and twodifferent heteroatom, e.g., N and O, O and S or N and S. Heterocyclicgroups include those having 5- or 6-member rings and a singleheteroatom, e.g., N S or O. Specific heterocyclic groups include amongothers among others, pyrrolidinyl, piperidyl, piperazinyl, pyrrolyl,pyrrolinyl, furyl, tetrahydropyranyl, tetrahydrofuryl, thienyl,morpholinyl, oxazolyl, oxazolinyl, oxazolidinyl, indolyl, triazoly, andtriazinyl groups, all of which are optionally substituted.

Aryl groups are monovalent groups containing at least one aromatic ring.Aryl groups include groups having one or more 5- or 6-member aromaticrings. Aryl groups can contain one, two or three, 6-member aromaticrings. Aryl groups can contain two or more fused aromatic rings. Arylgroups can contain two or three fused aromatic rings. Aryl groups maycontain one or more non-aromatic alicyclic rings in addition to anaromatic ring. Aryl groups are optionally substituted with one or morenon-hydrogen substituents as described herein. Substituted aryl groupsinclude among others those which are substituted with alkyl or alkenylgroups, which groups in turn can be optionally substituted. Specificaryl groups include phenyl groups, biphenyl groups, and naphthyl groups,all of which are optionally substituted as described herein. Substitutedaryl groups include fully halogenated or semihalogenated aryl groups,such as aryl groups having one or more hydrogens replaced with one ormore fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms.Substituted aryl groups include fully fluorinated or semifluorinatedaryl groups, such as aryl groups having one or more hydrogens replacedwith one or more fluorine atoms. Unless otherwise specified, an arylgroup can contain 5-20 carbon atoms or can contain 6-14 carbon atoms.Aryl groups also include those containing 6-12 carbon atoms.

Heteroaryl groups are monovalent groups having one or more aromaticrings in which at least one ring contains a heteroatom (a non-carbonring atom). Heteroaryl groups include those having one or twoheteroaromatic rings carrying 1, 2 or 3 heteroatoms and optionallyhaving one 6-member aromatic ring. Heteroaryl groups can contain 5-20,5-12 or 5-10 ring atoms. Heteroaryl groups include those having at leastone aromatic ring containing a heteroatom and one or two alicyclic,heterocyclic or aryl ring groups. Heteroaryl groups include those havingone aromatic ring containing a heteroatom and one aromatic ringcontaining carbon ring atoms. Heteroaryl groups include those having oneor more 5- or 6-member aromatic heteroaromatic rings and one or more6-member carbon aromatic rings. Heteroaromatic rings can include one ormore N, O, or S atoms in the ring. Heteroaromatic rings can includethose with one, two or three N, those with one or two O, and those withone or two S, or combinations of one or two or three N, O or S. Specificheteroaryl groups include pyridinyl, pyrazinyl, pyrimidinyl, quinolinyl,and purinyl groups.

Alkyl groups are monovalent groups and include straight-chain, branchedand cyclic alkyl groups. Unless otherwise indicated alkyl groups includethose having from 1 to 20 carbon atoms. Alkyl groups include alkylgroups having 1 to 3 carbon atoms, alkyl groups having from 4-7 carbonatoms and alkyl groups having 8 or more carbon atoms. Cyclic alkylgroups include those having one or more rings. Cyclic alkyl groupsinclude those which have 1, 2 or 3 rings. Cyclic alkyl groups alsoinclude those having 3-10 carbon atoms. Cyclic alkyl groups includethose having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring andparticularly those having a 3-, 4-, 5-, 6-, 7-, or 8-member ring. Thecarbon rings in cyclic alkyl groups can also carry straight-chain orbranched alkyl group substituents. Cyclic alkyl groups can includebicyclic and tricyclic alkyl groups. Alkyl groups are optionallysubstituted with one or more non-hydrogen substituents as describedherein. Specific alkyl groups include methyl, ethyl, n-propyl,iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl,n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl,cyclohexyl, decalinyl, and norbornyl, all of which are optionallysubstituted. Substituted alkyl groups include fully halogenated orsemihalogenated alkyl groups, such as alkyl groups having one or morehydrogens replaced with one or more fluorine atoms, chlorine atoms,bromine atoms and/or iodine atoms. Substituted alkyl groups includefully fluorinated or semifluorinated alkyl groups. Substituted alkylgroup include alkyl group substituted with one or more hydroxyl groups.Substituted alkyl groups include groups substituted with two or morehydroxyl groups, particularly where two hydroxyl groups are substitutedon adjacent carbon atoms.

Arylalkyl groups are monovalent alkyl groups substituted with one ormore aryl groups wherein the alkyl groups optionally carry additionalsubstituents and the aryl groups are optionally substituted. Specificarylakyl groups are phenyl-substituted alkyl groups, e.g., benzyl groupsor phenethyl groups which are optionally substituted. Heteroarylalkylgroups are monovalent alkyl groups substituted with one or moreheteroaryl groups wherein the alkyl groups optionally carry additionalsubstituents and the aryl groups are optionally substituted. Alkylarylgroups are monovalent aryl groups substituted with one or more alkylgroups wherein the alkyl groups optionally carry additional substituentsand the aryl groups are further optionally substituted. Specificalkylaryl groups are alkyl-substituted phenyl groups such as o-, m- orp-tolyl groups which are optionally substituted. Alkylheteroaryl groupsare monovalent alkyl groups substituted with one or more heteroarylgroups wherein the alkyl groups optionally carry additional substituentsand the heteroaryl groups are optionally substituted.

Alkenyl groups include monovalent straight-chain, branched and cyclicalkenyl groups which contain one or more carbon-carbon double bonds.Unless otherwise indicated alkenyl groups include those having from 2 to20 carbon atoms. Alkenyl groups include those having 2 to 4 carbon atomsand those having from 5-8 carbon atoms. Cyclic alkenyl groups includethose having one or more rings wherein at least one ring contains adouble bond. Cyclic alkenyl groups include those which have 1, 2 or 3rings wherein at least one ring contains a double bond. Cyclic alkenylgroups also include those having 3-10 carbon atoms. Cyclic alkenylgroups include those having a 5-, 6-, 7-, 8-, 9- or 10-member carbonring and particularly those having a 5- or 6-member ring. The carbonrings in cyclic alkenyl groups can also carry straight-chain or branchedalkyl or alkenyl group substituents. Cyclic alkenyl groups can includebicyclic and tricyclic alkyl groups wherein at least one ring contains adouble bond. Alkenyl groups are optionally substituted with one or morenon-hydrogen substituents as described herein. Specific alkenyl groupsinclude ethylene, propenyl, cyclopropenyl, butenyl, cyclobutenyl,pentenyl, pentadienyl, cyclopentenyl, cyclopentadienyl, hexylenyl,hexadienyl, cyclohexenyl, cyclohexadienyl, including all isomers thereofand all of which are optionally substituted. Substituted alkenyl groupsinclude fully halogenated or semihalogenated alkenyl groups.

Alkynyl groups include mono-valent straight-chain, branched and cyclicalkynyl group which contain one or more carbon-carbon triple bonds.Unless otherwise indicated alkynyl groups include those having from 2 to20 carbon atoms. Alkynyl groups include those having 2 to 4 carbon atomsand those having from 5-8 carbon atoms. Cyclic alkynyl groups includethose having one or more rings wherein at least one ring contains atriple bond. Cyclic alkynyl groups include those which have 1, 2 or 3rings wherein at least one ring contains a triple bond. Cyclic alkynylgroups also include those having 3-10 carbon atoms. Cyclic alkynylgroups include those having a 5-, 6-, 7-, 8-, 9- or 10-member carbonring and particularly those having a 5- or 6-member ring. The carbonrings in cyclic alkynyl groups can also carry straight-chain or branchedalkyl, alkenyl or alkynyl group substituents. Cyclic alkynyl groups caninclude bicyclic and tricyclic alkyl groups wherein at least one ringcontains a triple bond. Alkynyl groups are optionally substituted withone or more non-hydrogen substituents as described herein.

An alkoxy group is an alkyl group (including cycloalkyl), as broadlydiscussed above, linked to oxygen, a monovalent —O-alkyl group. Anaryloxy group is an aryl group, as discussed above, linked to an oxygen,a monovalent —O-aryl. A heteroaryloxy group is a heteroaryl group asdiscussed above linked to an oxygen, a monovalent —O-heteroaryl.Alkenoxy, alkynoxy, alicycloxy, heterocycloxy groups are analogouslydefined. All of such groups are optionally substituted.

As to any of the chemical groups herein which contain one or moresubstituents, it is understood, that such groups do not contain anysubstitution or substitution patterns which are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers arising from thesubstitution of these compounds.

The dithioamines of this invention as illustrated in formulas I, IA andIB can be prepared in view of the descriptions herein, what is known inthe art or by routine adaptation of art-known methods from startingmaterials and reagents which are commercially available or which can beprepared by methods that known in the art or routine adaptation of suchmethods. Kessler et al. (1994) [10a] and Servent et al. [10b] provideadditional methods for the synthesis of synthetic intermediates usefulfor preparation of the dithioamines of this invention.

DTBA was prepared via the two routes depicted in Scheme 1. A five-steproute commenced with the esterification of the amino acid and protectionof its amino group. Reduction with lithium aluminum hydride yielded adiol, which was subjected to Mitsunobu conditions to install therequisite sulfur functionality. [11] Deprotection gave DTBA as its HClsalt in 99% purity and an overall yield of 60%. A six-step route thatavoids generation of triphenylphosphine oxide, a recalcitrant byproductof the Mitsunobu reaction [11] provided DTBA.HCl in an overall yield of56%. In both routes, the product of every step is a white solid.

In a specific embodiment, dithioamines of this invention of formulas I,IA and IB in which R₁-R₃ and R₅ are hydrogens can be prepared employingmethods specifically described herein or routine adaptation of suchmethods employing 3-substitued derivatives of L-aspartic acid,D-aspartic acid, racemic aspartic acid and esters or amine-protectedderivatives thereof.

In an embodiment, the invention provides methods for synthesis of2-amino 1,4-dimercaptobutane and derivatives thereof of formulas I, IAand IB from aspartic acid and derivatives thereof as illustrated inScheme 1. An amino-protected diester of aspartic acid (e.g., compound 2)is reduced, for example with LiAlH₄, to the corresponding diol (e.g.,compound 3). The diol is then treated under Mitsunobu conditions withthioacetic acid nucleophile in the presence of an azodicarboxylatereagent, e.g., diisopropyl azodicarboxylate, or diethyl azodicarboxylateand trisubstituted phosphine, e.g., triphenylphospine, or tri (n-butyl)phosphine to form an acetylated dithiol (e.g., compound 4). Thisreaction is carried out between room temperature and 0° C., preferablyat 0° C., in anhydrous THF (polar aprotic solvent). Alternatively,dioxane or dichloromethane (DCM) can also be employed. The acetylateddithiol (e.g., compound 4) is then deacetylated and deprotected (ifdesired) to the dithiol amine (e.g., DTBA hydrochloride 5). The use ofthe phosphine can be avoided by reaction of the diol with a sulfonylchloride reagent, e.g., methanesulfonyl chloride or toluenesulfonylchloride, to form a disulfonate ester (e.g., compound 6). Thioacetate(CH₃CO-SH) is then used to displace the sulfonate in the presence ofcrown ether in polar aprotic solvent (e.g., DMF) and form the acetylateddithiol (e.g., compound 4), which in turn can be deacetylated to formthe dithioamine (e.g., compound 5). These methods can also be used toform S-acyl derivatives of formulas I, IA and IB where R₈ is other thanhydrogen by choice of thiocarboxylate (R₈CO—SH).

DTBA has desirable physicochemical attributes. Its HCl salt is a nearlyodorless white solid with high solubility in water. Using a pH-titrationmonitored by ultraviolet spectroscopy, [15] we determined the thiol pKavalues of DTBA be 8.2±0.2 and 9.3±0.1 (FIG. 3; Table 1) [16] Thesevalues are approximately 1 unit lower than those of DTT. This differenceis comparable to that between cysteamine and (WE, and likely resultsfrom the strong Coulombic and inductive effects of the protonated aminogroup. By equilibrating reduced DTBA with oxidized DTT and using HPLC toquantify reduced and oxidized species, we found the reduction potentialof oxidized DTBA to be E°′=(−0.317±0.002) V (FIG. 4; Table 1). This E°′value is slightly less than that of DTT, consistent with more acidicthiols forming less stable disulfide bonds [17] and with thepreorganization of DTT for disulfide-bond formation by its hydroxylgroups, which can form an intramolecular hydrogen bond and manifest agauche effect.

TABLE 1 Physical properties of disulfide-reducing agents. Thiol pK_(a)Disulfide Reduction Potential (E^(o)′) βME 9.61^(a) −0.260 V^(b)Cysteamine 8.37^(c) −0.203 V^(b) DTT (racemate) 9.2 (10.1)^(d) −0.327V^(e) DTBA 8.2 ± 0.2  (−0.317 ± 0.002) V^(f) (9.3 ± 0.1)^(f) ^(a)Valueis from ref. 12. ^(b)Values are from ref. 3f. ^(c)Values are from ref.13. ^(d)Values are from ref. 3a. ^(e)Value is from ref. 14. ^(f)Valuesare the mean ± SE from this work.

DTBA is an efficacious reducing agent for disulfide bonds in smallmolecules. We found that DTBA reduces the disulfide bond in oxidized [3ME 3.5-fold faster than does DTT at pH 7.0, and 4.4-fold faster at pH5.5 (FIG. 1A). These rate accelerations are commensurate with the lowerthiol pKa of DTBA. At pH 7.0, DTBA reduces oxidized L-glutathione5.2-fold more rapidly than does DTT (FIG. 1B). As oxidized L-glutathionehas a net charge of −2 near neutral pH, a favorable Coulombicinteraction could contribute to this higher rate acceleration.

DTBA is also an efficacious reducing agent for disulfide bonds inproteins. A cysteine residue resides within the active site of papain(Cys25) and near that of creatine kinase (Cys283). Forming a mixeddisulfide with that cysteine residue is known to eliminate theirenzymatic activities.[2c,18] These two enzymes differ, however, in theelectrostatic environment of their active sites. The active site ofpapain is hydrophobic like its substrates, though there is an anionicregion nearby (FIG. 2A).[19] In contrast, the active site of creatinekinase is cationic, complementary to its anionic substrates (FIG.2B).[20, 21a-c] DTBA reduces a disulfide bond in the hydrophobic/anionicactive site of papain 14-fold faster than does DTT (FIG. 2A). Incontrast, the two reagents reduce a disulfide bond near the cationicactive site of creatine kinase at a similar rate.

The amino group of DTBA confers additional benefits. For example, adisulfide-reducing agent that can be readily isolated, regenerated, andreused incurs less cost and generates less waste.[22] Moreover,extraneous disulfide bonds absorb light at 280 nm, which can confoundstandard measurements of protein concentration. [23] We reasoned thatDTBA could be isolated by its adsorption to a cation-exchange resin.Indeed, >99% of DTBA (but <1% of DTT) was removed from sodium phosphatebuffer, pH 8.0, upon addition of Dowex® 50 resin (see: The Examples). Wealso note that the amino group of DTBA enables its covalent attachmentto a soluble molecule, resin, or surface by simple reactions, such asreductive amination (which preserves the cationic charge) orN-acylation. We conclude that the attributes of DTBA enable it tosupplant DTT as the preferred reagent for reducing disulfide bonds inbiomolecules.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers, enantiomers, and diastereomers of the group members, aredisclosed separately. When a Markush group or other grouping is usedherein, all individual members of the group and all combinations andsubcombinations possible of the group are intended to be individuallyincluded in the disclosure. A number of specific groups of variabledefinitions have been described herein. It is intended that allcombinations and subcombinations of the specific groups of variabledefinitions are individually included in this disclosure. Compoundsdescribed herein may exist in one or more isomeric forms, e.g.,structural or optical isomers. When a compound is described herein suchthat a particular isomer, enantiomer or diastereomer of the compound isnot specified, for example, in a formula or in a chemical name, thatdescription is intended to include each isomers and enantiomer (e.g.,cis/trans isomers, R/S enantiomers) of the compound described individualor in any combination. Additionally, unless otherwise specified, allisotopic variants of compounds disclosed herein are intended to beencompassed by the disclosure. For example, it will be understood thatany one or more hydrogens in a molecule disclosed can be replaced withdeuterium or tritium. Isotopic variants of a molecule are generallyuseful as standards in assays for the molecule and in chemical andbiological research related to the molecule or its use. Isotopicvariants, including those carrying radioisotopes, may also be useful indiagnostic assays and in therapeutics. Methods for making such isotopicvariants are known in the art. Specific names of compounds are intendedto be exemplary, as it is known that one of ordinary skill in the artcan name the same compounds differently.

Molecules disclosed herein may contain one or more ionizable groups[groups from which a proton can be removed (e.g., —COON) or added (e.g.,amines) or which can be quaternized (e.g., amines)]. All possible ionicforms of such molecules and salts thereof are intended to be includedindividually in the disclosure herein. With regard to salts of thecompounds herein, one of ordinary skill in the art can select from amonga wide variety of available counterions those that are appropriate forpreparation of salts of this invention for a given application. Inspecific applications, the selection of a given anion or cation forpreparation of a salt may result in increased or decreased solubility ofthat salt.

Compounds of the invention, and salts thereof, may exist in theirtautomeric form, in which hydrogen atoms are transposed to other partsof the molecules and the chemical bonds between the atoms of themolecules are consequently rearranged. It should be understood that alltautomeric forms, that may exist, are included within the invention.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

Whenever a range is given in the specification, for example, atemperature range, a pH range, a time range, or a composition orconcentration range, all intermediate ranges and subranges, as well asall individual values included in the ranges given are intended to beincluded in the disclosure. It will be understood that any subranges orindividual values in a range or subrange that are included in thedescription herein can be excluded from the claims herein.

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art, insome cases as of their filing date, and it is intended that thisinformation can be employed herein, if needed, to exclude (for example,to disclaim) specific embodiments that are in the prior art. Forexample, when a compound is claimed, it should be understood thatcompounds known in the prior art, including certain compounds disclosedin the references disclosed herein (particularly in referenced patentdocuments), are not intended to be included in the claim.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting ^(of) excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. The broad termcomprising is intended to encompass the narrower consisting essentiallyof and the even narrower consisting of. Thus, in any recitation hereinof a phrase “comprising one or more claim element” (e.g., “comprising Aand B), the phrase is intended to encompass the narrower, for example,“consisting essentially of A and B” and “consisting of A and B.” Thus,the broader word “comprising” is intended to provide specific support ineach use herein for either “consisting essentially of” or “consistingof.” The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, catalysts, reagents, synthetic methods, purification methods,analytical methods, and assay methods, other than those specificallyexemplified can be employed in the practice of the invention withoutresort to undue experimentation. All art-known functional equivalents,of any such materials and methods are intended to be included in thisinvention. The terms and expressions which have been employed are usedas terms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed byexamples, preferred embodiments and optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims.

All references cited herein are hereby incorporated by reference to theextent that there is no inconsistency with the disclosure of thisspecification. Some references provided herein are incorporated byreference to provide details concerning sources of starting materials;alternative starting materials, reagents, methods of synthesis,purification methods, and methods of analysis; as well as additionaluses of the invention.

THE EXAMPLES Example 1 Materials and Methods

Commercial reagents were used without further purification.Dithiothreitol (DTT) was from Research Products International (Mt.Prospect, Ill.). Bis(2-mercaptoethyl)sulfone (BMS) was from Santa CruzBiotechnology (Santa Cruz, Calif.). Papain (lyophilized powder frompapaya latex), creatine kinase (lyophilized powder from rabbit muscle),hexokinase (lyophilized powder from Saccharomyces cerevisiae),glucose-6-phosphate dehydrogenase (ammonium sulfate suspension frombaker's yeast), N_(α)-benzoyl-L-arginine-4-nitroanilide hydrochloride,(S)-methyl methanethiosulfonate (Kenyon's reagent),trans-4,5-dihydroxy-1,2-dithiane (oxidized DTT), oxidized L-glutathione,oxidized 2-mercaptoethanol, and DOWEX 50WX4-400 ion-exchange resin werefrom Sigma Chemical (St. Louis, Mo.). Bis(2-mercaptoethyl) sulfonedisulfide (oxidized BMS) was synthesized as reported previously [2g]

All glassware was oven or flame-dried, and reactions were performedunder N₂(g) unless stated otherwise. Dichloromethane (DCM), diethylether, and tetrahydrofuran (THF) were dried over a column of alumina.Dimethylformamide (DMF) and triethylamine were dried over a column ofalumina and purified further by passage through an isocyanate scrubbingcolumn. Flash chromatography was performed with columns of 40-63 Åsilica, 230-400 mesh (Silicycle, Québec City, Canada). Thin-layerchromatography (TLC) was performed on plates of EMD 250-μm silica60-F₂₅₄. The term “concentrated under reduced pressure” refers to theremoval of solvents and other volatile materials using a rotaryevaporator at water aspirator pressure (<20 torr) while maintaining thewater-bath temperature below 40° C. Residual solvent was removed fromsamples at high vacuum (<0.1 torr). The term “high vacuum” refers tovacuum achieved by a mechanical belt-drive oil pump.

¹H NMR spectra were acquired at ambient temperature with a BrukerDMX-400 Avance spectrometer at the National Magnetic Resonance Facilityat Madison (NMRFAM) and referenced to TMS or residual protic solvent.¹³C NMR spectra were acquired with a Varian MercuryPlus 300 andreferenced to residual protic solvent. Electrospray ionization (ESI)mass spectrometry was performed with a Micromass LCT at the MassSpectrometry Facility in the Department of Chemistry at the Universityof Wisconsin-Madison. Ellman's assay for sulfhydryl groups was performedwith a Varian Cary 50 Bio UV-Vis spectrophotometer. UV absorbancespectra of oxidized DTBA and oxidized DTT were acquired with a VarianCary 300 Bio UV-Vis spectrophotometer. Thiol pK_(a) values weredetermined by using a Varian Cary 50 Bio UV-Vis spectrophotometer.Equilibrium, reduction potential, and kinetic studies on peptides andsmall molecules were performed on an analytical HPLC (Waters systemequipped with a Waters 996 photodiode array detector, Empower 2 softwareand a Varian C18 reverse phase column). Kinetic studies on proteins werecarried out using a Varian Cary 300 Bio UV-Vis spectrometer with a Carytemperature controller.

Example 2 Synthesis of DTBA

A.

L-Aspartic acid (1; 5.002 g, 37.58 mmol) was added to an oven-driedround-bottom flask and placed under an atmosphere of dry N₂(g). Thestarting material was then dissolved partially with 60 mL of anhydrousmethanol, and the mixture was cooled to 0° C. Once the desiredtemperature was reached, thionyl chloride (8.2 mL, 110 mmol) was addeddrop-wise. After the addition was complete, the reaction mixture becamehomogenous, and was warmed slowly to room temperature and left to stirfor 14 h. The reaction mixture was then concentrated under reducedpressure, and the resulting diester was dissolved in 150 mL of DCM and100 mL of water. To this biphasic solution was added sodium bicarbonate(4.212 g, 50.14 mmol) and di-t-butyl dicarbonate (9.841 g, 45.09 mmol),and the reaction mixture was heated at reflux for 4 h. After thereaction was confirmed to be complete by TLC, the reaction mixture wasallowed to cool to room temperature. The organic layer was separated,and the aqueous layer was extracted three times with 150 mL of DCM. Theorganic extracts were combined, washed with 250 mL of saturatedNaCl(aq), dried over MgSO₄(s), and concentrated under reduced pressure.Flash chromatography (35% v/v ethyl acetate in hexanes) was used toisolate 2 [(S)-dimethyl 2-(tert-butoxycarbonylamino)succinate] as awhite solid (9.080 g, 92%, 2 steps).

¹H NMR (400 MHz, CDCl₃) δ=5.49 (d, J=8.3 Hz, 1H), 4.60-4.57 (m, 1H),3.76 (s, 3H), 3.70 (s, 3H), 3.01 (dd, J=17, 4.4 Hz, 1H), 2.83 (dd,J=17.0, 4.7), 1.45 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ=171.6, 171.5,155.5, 80.3, 52.8, 52.1, 50.0, 36.8, 28.4; HRMS (ESI) calculated for[C₁₁H₁₉NO₆Na]⁺ (M+Na⁺) requires ink=284.1105, found 284.1113.

B.

An oven-dried round-bottom flask was charged with lithium aluminumhydride (0.870 g, 22.9 mmol) and placed under an atmosphere of dryN₂(g). The flask was cooled to 0° C. in an ice bath, and 100 mL ofanhydrous diethyl ether was added. In a separate dry round-bottom flask,compound 2 (2.021 g, 7.735 mmol) was dissolved in 50 mL of anhydrousdiethyl ether. Sonication was required to make the solution completelyhomogenous. The ester was then added drop-wise to the reaction mixture.Once the addition was complete, the reaction mixture was stirred at 0°C. for an additional 30 min, warmed to room temperature, and allowed toreact for an additional 2 h. Subsequently, the reaction mixture wasquenched at 0° C. by the slow, sequential addition of 0.87 mL of water,0.87 mL of 15% w/w NaOH, and 2.6 mL of water. The mixture was left tostir at room temperature for 1 h. The aluminum salts were collected byvacuum filtration, and subjected to continuous solid-liquid extractionswith dichloromethane using a Soxhlet apparatus. The organic extracts andthe original organic filtrate were combined and concentrated underreduced pressure. Flash chromatography (ethyl acetate) was used toisolate 3 [(S)-tert-butyl 1,4-dihydroxybutan-2-ylcarbamate] as a whitesolid (1.310 g, 82%). Compound 3 has been prepared from L-aspartic acidby a different route. [10a]

¹H NMR (400 MHz, DMSO-d₆) δ=6.46 (d, J=8.8 Hz, 1H), 4.56 (t, J=5.7 Hz,1H), 4.34 (t, J=5.1 Hz, 1H), 3.46-3.37 (m, 3H), 3.32 (dt, J=10.6, 5.4Hz, 1H), 3.23 (dt, J=10.6, 5.9 Hz, 1H), 1.69-1.61 (m, 1H), 1.45-1.37 (m,1H), 1.37 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ=157.2, 80.1, 65.4, 58.9,49.5, 35.0, 28.5; HRMS (ESI) calculated for [C₉H₁₉NO₄Na]⁺ (M+Na⁺)requires ink=228.1207, found 228.1201.

C.

A dry round-bottom flask was charged with triphenylphosphine (1.711 g,6.523 mmol) and placed under an atmosphere of dry N₂(g). Anhydrous THF(27 mL) was then added, and the solution was placed in an ice bath andcooled to 0° C. Diisopropyl azodicarboxylate (1.3 mL, 6.6 mmol) wasadded drop-wise to the flask. Once the addition was complete, thereaction mixture was allowed to stir for an additional 20 min. Compound3 (0.559 g, 2.72 mmol) in 10 mL of dry THF and thioacetic acid (0.47 mL,6.6 mmol) was then added with stirring. The reaction mixture was stirredat 0° C. for 1 h, and then at room temperature for 16 h. (Longerreaction times resulted in lower yields.) The mixture was concentratedunder reduced pressure. Flash chromatography (30% v/v ethyl acetate inhexanes) was used to isolate 4[(S)-S,S′-2-(tert-butoxycarbonylamino)butane-1,4-diyl diethanethioate]as a white solid (0.711 g, 81%). Compound 4 has been prepared formL-aspartic acid by a different route.[10a]

¹H NMR (400 MHz, CDCl₃) δ=4.59 (d, J=7.9 Hz, 1H), 3.85-3.76 (m, 1H),3.12-2.95 (m, 3H), 2.82 (ddd, J=13.7, 8.5, 7.1 Hz, 1H), 2.36 (s, 3H),2.33 (s, 3H), 1.84-1.75 (m, 1H), 1.74-1.64 (m, 1H), 1.44 (s, 9H); ¹³CNMR (75 MHz, CDCl₃) δ=195.9, 195.6, 155.6, 79.7, 50.1, 34.5, 33.8,30.73, 30.71, 28.5, 25.9; HRMS (ESI) calculated for [C₁₃H₂₃NO₄S₂Na]⁺(M+Na⁺) requires ink=344.0961, found 344.0962.

D.

A dry round-bottom flask was charged with 3 (1.178 g, 5.739 mmol) andplaced under dry N₂(g), Anhydrous DCM (125 mL) was then added, and thesolution was cooled to 0° C. Triethylamine (4.0 mL, 29 mmol) was added,followed by slow drop-wise addition of methanesulfonyl chloride (MsCl)(1.0 mL, 13 mmol). After stirring at 0° C. for 30 min, the reactionmixture was allowed to warm slowly to room temperature and left to reactfor an additional 30 min. The reaction mixture was quenched by theaddition 100 mL of water, and extracted with DCM. The combined organicextracts were washed with brine, dried over MgSO₄(s), and concentratedunder reduced pressure. Flash chromatography (60% v/v ethyl acetate inhexanes) was used to isolate 6[(S)-2-(tert-butoxycarbonyamino)butane-1,4-diyl dimethanesulfonate] as awhite solid (1.782 g, 86%).

¹H NMR (400 MHz, CDCl₃) δ=4.81 (d, J=9.7 Hz, 1H), 4.39-4.26 (m, 4H),4.10-4.05 (m, 1H), 3.06 (s, 3H), 3.05 (s, 3H), 2.13-1.96 (m, 2H), 1.48(s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ=155.4, 80.6, 71.0, 66.3, 47.0, 37.7,37.6, 31.2, 28.5; HRMS (ESI) calculated for [C₁₁H₂₃NO₈S₂Na]⁺ (M+Na⁺)requires ink=384.0758, found 384.0775.

E.

Compound 6 (0.610 g, 1.688 mmol), potassium thioacetate (0.482 g, 4.22mmol), and 18-crown-6 (1.351 g, 5.111 mmol) were added to a dryround-bottom flask and dissolved with 150 mL of anhydrous DMF. Thereaction mixture was stirred under dry N₂(g) for 24 h. The DMF wasremoved under reduced pressure. Flash chromatography (30% v/v ethylacetate in hexanes) was used to isolate 4[(S)—S,S′-2-(tert-butoxycarbonylamino)butane-1,4-diyl diethanethioate]as a white solid (0.475 g, 87%). Compound 4 has been prepared fromL-aspartic acid by a different route. [10a]

¹H NMR (400 MHz, CDCl₃) δ=4.59 (d, J=7.9 Hz, 1H), 3.85-3.76 (m, 1H),3.12-2.95 (m, 3H), 2.82 (ddd, J=13.7, 8.5, 7.1 Hz, 1H), 2.36 (s, 3H),2.33 (s, 3H), 1.84-1.75 (m, 1H), 1.74-1.64 (m, 1H), 1.44 (s, 9H); ¹³CNMR (75 MHz, CDCl₃) δ=195.9, 195.6, 155.6, 79.7, 50.1, 34.5, 33.8,30.73, 30.71, 28.5, 25.9; HRMS (ESI) calculated for [C₁₃H₂₃NO₄S₂Na]⁺(M+Na⁺) requires ink=344.0961, found 344.0962.

F.

Compound 4 (0.601 g, 1.87 mmol) was added to a flame-dried round-bottomflask under dry N₂(g). Anhydrous methanol (20 mL) was added, followed by10 mL of 3 N HCl in methanol. The reaction mixture was heated at refluxfor 4 h, concentrated under reduced pressure, and stored in vacuo withP₂O₅ and KOH for 48 h. [2a] (Scratching the bottom of the flaskfacilitated crystal formation.) Compound 5,(2S)-2-amino-1,4-dimercaptobutane hydrochloride, hereinS-dithiobutylamine hydrochloride (5-DTBA.HCl) was rinsed with coldtoluene, and isolated by vacuum filtration as a white solid (0.320 g,quant). S-DTBA made in this manner was determined to be 99% pureaccording to Ellman's assay for sulfhydryl groups (vide infra). [24]

¹H NMR (400 MHz, DMSO-d₆) δ=8.29 (s, 3H), 3.34-3.32 (m, 1H), 2.96 (t,J=8.7 Hz, 1H), 2.81-2.75 (m, 2H), 2.60-2.56 (m, 3H), 1.95-1.86 (m, 2H);¹³C NMR (75 MHz, DMSO-d₆) δ=51.2, 35.0, 26.0, 19.6; HRMS (ESI)calculated for [C₄H₁₂NS₂]⁺ (M⁺) requires m/z=138.0406, found 138.0405.

G.

Compound 4 (0.482 g, 1.50 mmol) and potassium hydroxide (0.340 g, 6.06mmol) were dissolved in 50 mL of methanol, and the resulting solutionwas stirred for 16 h while bubbling a light stream of air through thesolution. The methanol was removed under reduced pressure, and themixture was extracted with DCM, washed with brine, and dried overMgSO₄(s). Flash chromatography (20% v/v ethyl acetate in hexanes) wasused to isolate 7 [(S)-tert-butyl 1,2-dithian-4-ylcarbamate] as a whitesolid (0.331 g, 94%).

¹H NMR (400 MHz, DMSO-d₆) δ=7.08 (d, J=7.9 Hz, 1H), 3.53-3.41 (m, 1H),3.07-3.01 (m, 1H), 2.91-2.85 (m, 2H), 2.60 (dd, J=13.0, 10.5 Hz, 1H),2.08-2.03 (m, 1H), 1.67-1.57 (m, 1H), 1.38 (s, 9H); ¹³C NMR (75 MHz,DMSO-d₆) δ=155.2, 78.7, 49.3, 37.9, 34.9, 34.5, 28.9; HRMS (ESI)calculated for [C₉H₁₇NO₂S₂]⁺ (M⁺) requires m/z=258.0593, found 258.0602.

F.

Compound 7 (0.402 g, 1.71 mmol) was added to a round-bottom flask.Anhydrous methanol (20 mL) was added, followed by 10 mL of 3 N HCl inmethanol. The reaction mixture was heated at reflux for 4 h under N₂(g),concentrated under reduced pressure, and stored in vacuo with P₂O₅ andKOH for 24 h. (Scratching the bottom of the flask facilitated crystalformation.) Compound 8, oxidized DTBA.HCl [(S)-1,2-dithian-4-aminehydrochloride], was isolated as a white solid (0.289 g, quant).

¹H NMR (400 MHz, DMSO-d₆) δ=8.29 (s, 3H), 3.43-3.37 (m, 1H), 3.15-3.08(m, 2H), 3.02-2.96 (m, 1H), 2.88 (dd, J=13.1, 10.6 Hz, 1H), 2.32-2.28(m, 1H), 1.85-1.77 (m, 1H); ¹³C NMR (75 MHz, DMSO-d₆) δ=48.7, 34.6,32.8, 31.5; HRMS (ESI) calculated for [C₄H₁₀NS₂]⁺ (M⁺) requiresm/z=136.0250, found 136.0249.

Example 3 Purity of DTBA Assessed by Ellman's Assay for SulfhydrylGroups

A reaction buffer (0.10 M sodium phosphate buffer, pH 8.0, containing 1mM EDTA) was prepared by the Pierce protocol. Ellman's reagent solutionwas primed by adding Ellman's reagent (4 mg) to 1 mL of the reactionbuffer. A 2.50×10⁻⁴ M solution of DTBA was then prepared using thereaction buffer. Ellman's reagent solution (50 μL) was added to each oftwo vials containing 2.5 mL of reaction buffer. Reaction buffer (250 μL)was added to one of these vials, and its absorbance at 412 nm was usedas a blank. DTBA solution (250 μL) was added to the other vial. After 10min, its absorbance at 412 nm was recorded. Using Beer's law (c=A/(ε·l)with A=0.623, l=1 cm, and ε=14,150 M⁻¹cm⁻¹) gave a thiol concentrationof 4.40×10⁻⁵ M. Because DTBA contains two thiol groups, the assaysolution had a DTBA concentration of 2.20×10⁻⁵ M. Accounting fordilution and using the equation M₁·V₁=M₂·V₂, where V₁=2.50×10⁻⁴ L,M₂=2.20×10⁻⁵ M, and V₂=2.8×10⁻³ L, yielded M₁=2.46×10⁻⁴ M and thus aDTBA purity of (2.46×10⁻⁴ M)/(2.50×10⁻⁴ M)×100%=98.4%. Three repetitionsof this assay gave (99±1)% purity. This assay revealed that commercialDTT and BMS had >98% purity.

Example 4 Determination of Thiol pK_(a) Values

The thiol pK_(a) values of DTBA were determined by measuring itsabsorbance at 238 nm in solutions of different pH. The deprotonatedthiolate absorbs much more strongly at 238 nm than does its protonatedcounterpart. [15a] This attribute was exploited for determining thiolpK_(a) values as described previously. [15b] Buffered stock solutions ofK₃PO₄, K₂HPO₄, and KH₂PO₄ (100 mM) were degassed and flushed with N₂(g)for 1 h immediately prior to use. A stock solution of DTBA (1.5 mM) inKH₂PO₄ was then prepared. Various combinations of the buffered stocksolutions were combined in duplicate to give two identical sets of 1-mLsolutions of pH 5.5-11. KH₂PO₄ stock solution (70 μL) was added to eachreplicate pair of solutions and used to set the A₂₃₈ to zero. Dithiolsolution (70 μL) was then added to its complimentary 1-mL vial, and itsabsorbance at 238 nm was recorded. The pH of the solution was thenimmediately measured using a Beckman pH meter, which had been calibratedprior to use with pH 7 and pH 10 standard solutions from FisherScientific. This process was repeated multiple times to obtain a plot ofA₂₃₈ vs pH (FIG. 3).

pK_(a) values were determined by fitting the data in FIG. 3 to eq 1,which is derived from Beer's law and the definition of the aciddissociation constant. [15b] In eq 1, C_(T) is total thiolconcentration, ε_(SH) ^(SH) is the extinction coefficient of the doublyprotonated form, ε_(SH) ^(S−) is the extinction coefficient of thesingly protonated form, and ε_(S−) ^(S−)is the extinction coefficient ofthe unprotonated form. Both pK_(a) values and extinction coefficientswere determined from the curve fit.

$\begin{matrix}{A_{238} = {C_{T}\left( \frac{{ɛ_{S^{-}}^{S^{-}}10^{{p\; H} - {p\; K_{a\; 2}}}} + ɛ_{SH}^{S^{-}} + {ɛ_{SH}^{SH}10^{{p\; K_{a\; 1}} - {p\; H}}}}{10^{{p\; H} - {p\; K_{a\; 2}}} + 1 + 10^{{p\; K_{a\; 1}} - {p\; H}}} \right)}} & (1)\end{matrix}$

Example 5 Reduction Potential of DTBA

The reduction potential (E°′) of DTBA was determined by using HPLC todetermine the equilibrium constant for its reaction with oxidized DTT(eq 2), and then inserting this value into a variation of the Nernstequation (eq 3). [2g] Data were obtained by a procedure similar to thatdescribed previously. [2g, 15b] DTBA (10.5 mg, 0.06 mmol) and oxidizedDTT (9.2 mg, 0.06 mmol) were added to a 25-mL round-bottom flask. Theflask was then flushed with N₂(g) for 30 min.

$\begin{matrix}{K_{eq} = {\frac{\lbrack{DTT}\rbrack \left\lbrack {{oxidized}\mspace{14mu} {DTBA}} \right\rbrack}{\lbrack{DTBA}\rbrack \left\lbrack {{oxidized}\mspace{14mu} {DTT}} \right\rbrack} = \frac{\lbrack{DTT}\rbrack^{2}}{\left\lbrack {{oxidized}\mspace{14mu} {DTT}} \right\rbrack^{2}}}} & (2) \\{E_{DTBA}^{o^{\prime}} = {E_{DTT}^{o^{\prime}} - {\frac{RT}{n\; F}\ln \frac{\; \lbrack{DTT}\rbrack^{2}}{\left\lbrack {{oxidized}\mspace{14mu} {DTT}} \right\rbrack^{2}}}}} & (3)\end{matrix}$

A 50 mM stock solution of potassium phosphate buffer (pH 7) was degassedand purged with N₂(g) for 30 min immediately prior to use. Buffer (15mL) was added, and the reaction mixture was stirred under N₂(g) for 24 hat room temperature. The reaction mixture was then quenched by theaddition of 3 N HCl (1:100 dilution). The reaction mixture was passedthrough a 4.5-μm filter, and 100 μL of the reaction mixture was analyzedimmediately by HPLC using a Waters system equipped with a Waters 996photodiode array detector, Empower 2 software, and a Varian C18reverse-phase column. The column was eluted at 1.0 mL/min with water(5.0 mL), followed by a linear gradient (0-40% v/v) ofacetonitrile/water over 40 min. Compounds were detected by theirabsorbance at 205 nm. Reduced and oxidized DTBA are highly polar andelute from the column immediately (as confirmed by LC-MS). Two peaks,however, were clearly visible in the chromatogram FIG. 4). HPLC analysisof standards revealed that the two peaks were reduced DTT (retentiontime: 19 min) and oxidized DTT (retention time: 23 min). Calibrationcurves were generated and found to be linear over the used concentrationrange. From these curves, the equilibrium concentrations of reduced andoxidized DTT were determined, and a K_(eq)=0.469±0.131 for the reactionwas found. Assuming that DTT has E°′=−0.327 V, [2a] eq 3 (which is avariation of the Nernst equation) was used to calculate that DTBA hasE°′=−(0.317±0.002) V. This value is the mean±SE from seven experiments.The reverse reaction between oxidized DTBA and reduced DTT revealed thatequilibrium had been established under the experimental conditions.

Example 6 Reduction Potential of BMS

The procedure described in Example 5 was also performed with BMS. WithK_(eq)=0.0517±0.0194 and assuming E°′=−0.327 V for DTT, [2a] BMS wasfound to have E°′=(−0.291±0.002) V, which was again the mean±SE fromseven experiments. A previously reported reduction potential for BMS wasE°′=−0.31 V. [2g]

Example 7 Kinetic Studies on the Reduction of Oxidized βME

${- \frac{\partial\lbrack{disulfide}\rbrack_{total}}{\partial t}} = {{k_{obs}\lbrack{disulfide}\rbrack}_{total}\lbrack{thiol}\rbrack}_{total}$

The observed second-order rate constant (k_(obs)) for a thiol-disulfideinterchange reaction was determined by adapting a procedure describedpreviously. [2e] When the disulfide is oxidized βME, a 50 mM stocksolution of potassium phosphate buffer was degassed and purged withN₂(g) for 30 min immediately prior to use. A stock solution of oxidizedβME (10 mM) in 50 mM potassium phosphate buffer, pH 7.0, was purged withN₂(g) for 30 min immediately prior to use. A 25-mL round-bottom flaskwas charged with DTBA (4.3 mg, 0.025 mmol) or DTT (3.9 mg, 0.025 mmol),and placed under N₂(g). Phosphate buffer (2.5 mL) was added to theround-bottom flask containing the dithiol. Oxidized βME stock solution(2.5 mL) was then added, and the reaction mixture was stirred at roomtemperature under N₂(g) for 1 min. The reaction mixture was quenched bythe addition of 0.10 mL of 3 N HCl. The reaction mixture was passedthrough a 4.5-μm filter, and 100 μL of the reaction mixture was analyzedimmediately by HPLC using a Varian C18 reverse-phase column. The columnwas eluted at 1.0 mL/min with water (5.0 mL), followed by a lineargradient (0-40% v/v) of acetonitrile/water over 40 min. The extent ofreduction was determined by integrating the newly formed peakcorresponding to βME at 205 nm (retention time: 8 min). This process wasrepeated for reaction times of 2 and 4 min. Calibration curves weregenerated and found to be linear over the used concentration range. Theamount of residual oxidized βME was calculated, and second-order rateconstants were calculated from a linear fit of the data in FIG. 1A (thatis, k_(obs)=[(1/c_(final))−(1/c_(initial))]/t). The initial values ofconcentration in the reaction mixture were [DTBA or DTT]=[oxidizedβME]=c_(intitial)=5 mM. Rate constants were the mean±SE from threeexperiments. DTBA: k_(obs)=(0.29±0.02) M⁻¹s⁻¹ and DTT:k_(obs)=(0.084±0.004) M⁻¹s⁻¹. The same procedure was performed forreactions at pH 5.5, giving DTBA: k_(obs)=(0.0093±0.0003) M⁻¹s⁻¹ andDTT: k_(obs)=(0.0021±0.0002) M⁻¹s⁻¹ (FIG. 1A).

Example 8 Kinetic Studies on the Reduction of Oxidized L-Glutathione

An experiment similar to that in Example 7 was conducted withdisulfide=oxidized L-glutathione. Reactions were quenched at varioustime points (2, 4, 6, and 8 min) and 100 μL was analyzed by HPLC (1.0mL/min with water (5.0 mL) in 0.1% v/v TFA, followed by a lineargradient (0-40% v/v) of acetonitrile in 0.1% v/v TFA over 40 min). Theextent of reduction was determined by integrating the newly formedL-glutathione reduced peak at 220 nm (retention time of 7 min).Second-order rate constants were calculated from a linear fit of thedata in FIG. 1B (that is, k_(obs)=[(1/c_(final))−(1/c_(initial))]/t).Rate constants were the mean±SE from three experiments. DTBA:k_(obs)=(0.83±0.04) M⁻¹s⁻¹ and DTT: k_(obs)=(0.16±0.02) M⁻¹s⁻¹.

Example 9 Kinetic Studies on the Reactivation of Papain

Cys25 in the active site of papaya latex papain was oxidized as a mixeddisulfide by a procedure described previously.[25] Briefly, a stocksolution of methyl methane-thiosulfonate (3.5 mM) was prepared bydilution of 5 μL of methyl methanethiosulfonate with 15 mL of 0.10 Mpotassium phosphate buffer, pH 7.0, containing EDTA (2 mM). KCl (0.011g, 0.15 mmol) was added to 1.5 mL of this stock solution. The solutionwas deoxygenated by bubbling N₂(g) through it for 15 min. Next, papain(5 mg, 150 units) was added, and the resulting solution was incubated atroom temperature under N₂(g) for 12 h. Excess methylmethanethiosulfonate was removed by size-exclusion chromatography usinga Sephadex G-25 column. The final concentration of papain was determinedby A₂₈₀ using ε₂₈₀=5.60×10⁴ M⁻¹cm⁻¹. [26] A solution (0.26 mL) of thechromatographed protein was diluted with 4.94 mL of deoxygenated aqueousbuffer (0.10 M imidazole-HCl buffer, pH 7.0, containing 2 mM EDTA).Enzyme solution (1.25 mL) was then added to four separate vials. DTBA orDTT (10 μL of a 1 mM solution) was added to one of the vials, and atimer was started. The initial concentrations in the reaction mixturewere dithiol reducing agent: 7.9×10⁻⁶ M and inactive protein: 4.9×10⁻⁶M. At various times, an 0.20-mL aliquot was removed from the reactionmixture and added to a cuvette of 0.8 mL of substrate solution (1.25 mMN-benzoyl-L-arginyl-p-nitroanilide in 0.10 M imidazole-HCl buffer, pH6.0, containing 2 mM EDTA). The rate of change in absorbance at 410 nmwas recorded at 25° C. A unit of protein is defined by the amount ofenzyme required to produce 1 μmol/min of 4-nitroaniline. Using anextinction coefficient for 4-nitroaniline of ε=8,800 M⁻¹cm⁻¹ at 410 nm,[27] the number of units of active papain in solution at each time pointwas calculated. To determine the possible number of units of activepapain in the reaction mixture, a large excess of DTT (˜10³-fold) wasadded to one vial and the activity was assessed. As a control, it wasdetermined that the concentrations of DTT used had no bearing on theassay data other than activating the protein. Enzymatic activity (%) atparticular times was calculated by dividing the number of active unitsof enzyme by the possible number of units in the solution, and wasplotted as in FIG. 2A. To determine the value of the second-order rateconstant k_(obs) for the reducing agents, the second-order rate equation(eq 4) was transformed into eq 5, which was fitted to the data with theprogram PRISM 5.0. In eq 4 and 5, A₀=[inactive protein]_(t=0),A=[inactive protein]_(t)=A₀-A₀y, B₀=[reducing agent]_(t=0), andB=[reducing agent]_(t)=B₀-A₀y. Values of k_(obs) were the mean±SE fromthree experiments. DTBA: k_(obs)=(1342±148) M⁻¹s⁻¹ and DTT:k_(obs)=(91.3±9.4) M⁻¹s⁻¹.

$\begin{matrix}{{\frac{1}{B_{0} - A_{0}}\ln \frac{\; {A_{0}B}}{{AB}_{0}}} = {k_{obs}t}} & (4) \\{y = {\frac{B_{o} - {B_{o}^{k_{obs}{t{({A_{o} - B_{o}})}}}}}{B_{o} - {A_{o}^{k_{obs}{t{({A_{o} - B_{o}})}}}}} \times 100\%}} & (5)\end{matrix}$

Example 10 Kinetic Studies on the Reactivation of Creatine Kinase

Cys283 in the active site of rabbit muscle creatine kinase was oxidizedas a mixed disulfide by a procedure described previously, [28] but witha slight modification in the measurement of active enzyme. A unit ofenzyme was defined as the amount required to produce 1 μmol/min ofNADPH. Using an extinction coefficient for NADPH of 68 =6.22 mM⁻cm⁻¹ at340 nm, the units of active creatine kinase in solution at a particulartime were calculated. To determine the possible number of units ofactive creatine kinase in the reaction mixture, a large excess of DTT(˜10³-fold) was added to one vial and the activity was assessed. As acontrol, it was determined that the concentrations of DTT used had nobearing on the assay data other than activating the protein. Enzymaticactivity (%) at particular times was calculated by dividing the numberof active units of enzyme by the possible number of units in thesolution, and was plotted as in FIG. 2B. Values of the second-order rateconstant k were determined by using eq 4 as described in Example 9, andwere the mean±SE from three experiments. DTBA: k_(obs)=(15.1±1.0) M⁻s⁻¹and DTT: k_(obs)=(17.5±1.6) M⁻¹s⁻¹.

Example 11 Separation of DTBA Using an Ion-Exchange Resin

A reaction buffer (0.10 M sodium phosphate, pH 8.0, 1 mM EDTA) wasprepared. Ellman's reagent solution was prepared by adding Ellman'sreagent (4 mg) to 1 mL of the reaction buffer. Next, to 25 mL ofreaction buffer (0.10 M sodium phosphate, pH 8.0, 1 mM EDTA) was addedDTBA (2.2 mg, 1.27×10⁻⁵ mol) and 1.7 g of DOWEX 50WX4-400 ion-exchangeresin. The mixture was swirled for several minutes and filtered througha fritted syringe. Ellman's reagent solution (50 μL) was added to twoseparate vials containing 2.5 mL of reaction buffer. As a blank, 250 μLof reaction buffer was added to one of the vials, and the absorbance at412 nm was set to zero. Filtrate (250 μL) was then added to the othervial and its absorbance was recorded. With A₄₁₂=0.012 and using anextinction coefficient of ε=14,150 M⁻¹cm⁻¹, [24b, 24c] it was calculatedthat >99% of DTBA was retained by the resin and thus removed fromsolution. The same assay was repeated with DTT, resulting in <1% beingremoved from solution. See Example 3 for a more detailed explanation ofsimilar calculations using Ellman's assay.

Example 12 Ultraviolet Spectra of Oxidized DTBA and Oxidized DTT

Solutions of oxidized DTBA and DTT (1.0 mM) were prepared in Dulbecco'sphosphate buffered saline (DPBS), and their ultraviolet spectra wererecorded (FIG. 6).

Example 13 Preparation of Immobilized Dithiobutylamine (DTBA)

A. Coupling of DTBA^(ox) to Preactivated TentaGel® Resin

Preactivated succinimidyl ester resin (1 g, ˜0.21 mmol/g) with aparticle size of 130 μm and capacity of 0.2-0.3 mmol/g (TentaGel®COOSu,Rapp Polymere GmbH, Tuebingen, Germany) was placed in a solid phasepeptide synthesis vessel. As a pretreatment, the resin was allowed toswell in 5 mL of N-methyl-2-pyrrolidone (NMP) for five min. whilebubbling nitrogen through the solution. The NMP was then removed byvacuum filtration and the process was repeated two more times. The resinused in this example is a grafted copolymer with a cross-linkerpolystyrene matrix on which polyethylene glycol (PEG or POE) is graftedand the PEG spacer is in the range of MW 3000 Da. To the resin was thenof added 10 mL of NMP, 0.320 mL (1.8 mmol) of N,N-diisopropylethylamine(DIEA), and 0.1224 g (0.7128 mmol) of compound 8 (DTBA^(ox)). Theresulting mixture was allowed to react for 60 min while bubblingnitrogen through the solution. The solution was then removed by vacuumfiltration and the resin was washed three times with 5 mL of NMP. In asecond coupling step, 10 mL of NMP, 0.470 g (0.903 mmol) ofBenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), 0.320 mL (1.8 mmol) of DIEA and 0.1224 mg (0.7128 mmol) ofcompound 8 were added to the resin and allowed to react for anadditional hour while bubbling nitrogen through the solution. Thesolution was then removed by vacuum filtration.

B. Reduction/Regeneration of Immobilized DTBA^(ox) (3 Methods)

To 1 g of immobilized DTBA^(ox) (108), in a solid phase peptidesynthesis vessel, was added 0.260 g (0.907 mmol) oftris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) in a 10 mL of 50mM phosphate buffer, pH 6. The mixture was allowed to react for 60 minwhile bubbling nitrogen gas through the solution. The solution was thenremoved by vacuum filtration, and the resin was washed 5 times with 5 mLof 0.1 M acetic acid to ensure the thiol groups were protonatedcompletely to produce immobilized DTBA (110). The resin was then washed3 times with 5 mL of methanol, and dried under vacuum overnight.

To 1 g of immobilized DTBA^(ox) (108), in a solid-phase peptidesynthesis vessel, was added 0.551 g (2.1 mmol) of triphenylphosphine(TPP) in 10 mL of H₂O/THF (80:20). The mixture was allowed to react for60 min while bubbling nitrogen gas through the solution. The solutionwas then removed by vacuum filtration, and the resin was washed 5 timeswith 5 mL of 0.1 M acetic acid to ensure the thiol groups werecompletely protonated to generate immobilized DTBA (110). The resin wasthen washed 3 times with 5 mL of methanol, and dried under vacuumovernight.

To 1 g immobilized DTBA^(ox) (108), in a solid phase peptide synthesisvessel, was added 79.4 mg (2.10 mmol) of NaBH₄ in 10 mL of methanol.Nitrogen has was bubbled through the solution, while the mixture wasallowed to react. After 60 min, the solution was removed by vacuumfiltration and washed 5 times with 5 mL of 0.1 M acetic acid to ensurethe thiol groups were completely protonated to generate immobilized DBTA(110). The resin was then washed 3 times with 5 mL of methanol, anddried under vacuum overnight.

The above procedures were performed multiple times with yields ofimmobilized DTBA ranging from 75-93%, as determined by Ellman's assayfor sulfhydryl groups.

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We claim:
 1. A compound having formula I or II:

or salts thereof where: R₁ is hydrogen, or an unsubstituted alkyl grouphaving 1 to 3 carbon atoms; each R₂ and R₃ is independently hydrogen, analkyl group having 1-3 carbon atoms, a phenyl or a benzyl group each ofwhich is optionally substituted with one or more of substituents W₂;each R₄ and R₅ is independently hydrogen, a halogen, a cyano group, anitro group, a hydroxyl, an alkyl group having 1-6 carbon atoms, aphenyl, a benzyl group, an N(R₉)₂, or a —COR₁₀ group, wherein eachalkyl, phenyl, or benzyl group is optionally substituted with one ormore of substituents W₂; each R₆ and R₇, is independently hydrogen, a1-12 carbon alkyl group, an aryl group, a heterocyclic group, aheteroaryl group, a —COR₁₁ group, a —COOR₁₁ group, a —CO—NHR₁₁ group, a—CO—NHR₁₁ group, a —SO₂—R₁₁ group, or a —(CH₂)_(n)—R₁₂ group, whereineach alkyl, aryl heterocylic or heteroaryl group is optionallysubstituted with one or more of substituents W₃; and each R₈ isindependently hydrogen or —CO—R₁₃, wherein: each R₉ is independentlyhydrogen, a 1-12 carbon alkyl group, an aryl group, a heterocyclicgroup, a heteroaryl group, a —COR₁₁ group, a —COOR₁₁ group, a —CO—NHR₁₁group, a —CO—NHR₁₁ group, a —SO₂—R₁₁ group, or a —(CH₂)n-R₁₂ group wheren is an integer ranging from 1-12, wherein each alkyl, aryl heterocyclicor heteroaryl group is optionally substituted with one or more ofsubstituents W₃; each R₁₀ is independently hydrogen, a 1-12 alkyl group,a phenyl or benzyl group which are optionally substituted with one ormore of substituents W₂; each R₁₁ and R₁₂ is independently hydrogen, a1-12 carbon alkyl group, an aryl group, a heterocyclic group, aheteroaryl group, a -L-T group or a -M group, wherein each alkyl, arylheterocylic or heteroaryl group is optionally substituted with one ormore of substituents W₃, -L- is a divalent linker group and T is abiological species or a surface to which the reducing agent is linkedand -M is a reactive group or a spacer moiety carrying a reactive group;and each R₁₃ is independently hydrogen, a 1-12 carbon alkyl group, anaryl group, a heterocyclic group, or a heteroaryl group, wherein eachalkyl, aryl heterocyclic or heteroaryl group is optionally substitutedwith one or more of substituents W₃; wherein: W₂ is one or moresubstituents selected from halogen, an oxo group (═O), cyano group,nitro group, hydroxyl, unsubstituted alkyl group having 1-3 carbonatoms, halogen-substituted alkyl group having 1-3 carbon atoms, orunsubstituted alkoxy group having 1-3 carbon atoms; and W₃ is one ormore substituents selected from halogen, an oxo group (═O), cyano group,nitro group, hydroxyl, optionally substituted alkyl group having 1-6carbon atoms, unsubstituted alkyl group having 1-6 carbon atoms,hydroxyl-substituted alkyl group having 1-6 carbon atoms,halogen-substituted alkyl group having 1-6 carbon atoms, unsubstitutedalkoxy group having 1-6 carbon atoms, alkenyl group having 2-6 carbonatoms; alkynyl group having 2-6 carbon atoms, a 3-6-member alicyclicring, wherein one or two ring carbons are optionally replaced with —CO—and which may contain one or two double bonds, an aryl group having 6-14carbon ring atoms, a phenyl group, a benzyl group, a 5- or 6-member ringheterocyclic group having 1-3 heteroatoms and wherein one or two ringcarbons are optionally replaced with —CO— and which may contain one ortwo double bonds, or a heteroaryl group having 1-3 heteroatoms (N, O orS), —CO₂R₁₄ group, —CON(R₁₅)₂ group, —OCON(R₁₅)₂ group, —N(R₁₅)₂ group,a —SO₂—OR₁₅ group, —(CH₂)_(m)—OR₁₄ group, —(CH₂)_(m)—N(R₁₅)₂, where m is1-8, and each R₁₄ and R₁₅ is independently hydrogen, an unsubstitutedalkyl group having 1-6 carbon atoms; an unsubstituted aryl group having6-14 carbon atoms, an unsubstituted phenyl group; an unsubstitutedbenzyl group, an unsubstituted 5- or 6-member ring heterocyclic group,having 1-3 heteroatoms and wherein one or two ring carbons areoptionally replaced with —CO— and which may contain one or two doublebonds, or a unsubstituted heteroaryl group having 1-3 heteroatoms, withthe exception that no R₁₄ is hydrogen, wherein in the compound offormula, at least one of R₁-R₇ is a group other than a hydrogen.
 2. Thecompound of claim 1 wherein neither R₄ or R₅ is an —NH₂ group.
 3. Thecompound of claim 1 wherein R₁, R₂ and R₃ are all hydrogens.
 4. Thecompound of claim 1 wherein R₁, R₂, R₃, R₄ and R₅ are all hydrogens. 5.The compound of claim 1 wherein both R₈ are hydrogens.
 6. The compoundof claim 1 wherein both of R₈ are acyl groups.
 7. The compound of claim1 wherein one of R₆ or R₇ is an acyl group.
 8. The compound of claim 1wherein both of R₆ or R₇ are hydrogens.
 9. The compound of claim 1wherein R₁₁ or R₁₂ is -M.
 10. The compound of claim 1 wherein R₁₁ or R₁₂is -L-T.
 11. The compound of claim 1 wherein R₁-R₈ are all hydrogens.12. The compound of claim 1 wherein R₁-R₇ are all hydrogens and each R₈is a —CO—R₁₃ wherein R₁₃ is an alkyl group having 1-6 carbon atoms, ahalogen-substituted alkyl group having 1-6 carbon atoms, ahydroxy-substituted alkyl group having 1 to 6 carbon atoms, anoptionally substituted phenyl group or an optionally substituted benzylgroup.
 13. The compound of claim 1 wherein R₁, R₂, R₃ and one of R₄ orR₅ are all hydrogens and the other of R₄ or R₅ is an optionallysubstituted alkyl group having 1-3 carbons atoms, a halogen, a hydroxyl,or an acyl group.
 14. The compound of claim 1 having formula I.
 15. Thecompound of claim 1 which is non-racemic having formula:


16. The compound of claim 15 wherein R₁-R₃ are all hydrogens.
 17. Thecompound of claim 15 wherein R₁-R₇ are all hydrogens.
 18. The compoundof claim 15 wherein R₈ are both hydrogens.
 19. The compound of claim 15wherein R₈ are both acyl groups.
 20. The compound of claim 1 which iscovalently conjugated to a surface.
 21. The compound of claim 1 which iscovalently conjugated to a peptide, protein, carbohydrate or a nucleicacid.
 22. The compound of claim 1 is covalently conjugated to a ligandwhich binds to a peptide or protein or a substrate of an enzyme.
 23. Amethod for reducing or preventing disulfide bond formation in one ormore molecules having one or more sulfhydryl groups which comprises thestep of contacting the one or more molecules with one or more compoundsof formula I of claim 1
 24. The method of claim 23 wherein thecontacting is carried out at a pH ranging from 6-8.
 25. The method ofclaim 23 wherein the contacting is carried out at a pH ranging from 6.5to 7.5.
 26. The method of claim 23 wherein the one or more compounds arecovalently attached to a surface.
 27. The method of claim 23 whereinboth R₈ of the one or more compounds are acyl groups and the acyl groupsof the compound are removed prior to or at the same time as thecontacting.
 28. The method of claim 22 wherein reducing or preventingdisulfide bond formation reduces or prevents the formation of dimers orother oligomers of the one or more molecules having sulfhydryl groups.29. The method of claim 22 wherein the molecules having one or moresulfhydryl groups are peptides or proteins and reducing or preventingdisulfide bond formation functions to modulate a biological activity ofthe one or more peptides or proteins.
 30. A kit comprising one or moreof the compounds of claim 1 in combination with one or more componentsselected from one or more molecules having one or more sulfhydrylgroups, or one or more solvents or buffers for the one or more moleculesor one or more compounds.